Sample preparation methods, systems and compositions

ABSTRACT

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/484,856, filed Apr. 12, 2017 which is incorporated herein byreference in its entirety.

BACKGROUND

The analysis of genetic material in a sample has numerous potential usesand applications, including the identification of genetic indicators ofdisease (e.g., cancer), infection, disease progression, and fetalhealth. Advances in high-throughput sequencing technologies andPCR-based approaches have permitted more accurate identification of suchgenetic material. Before these approaches can be used, usually astarting sample is processed in some manner. For example, nucleic acidsmay be extracted or purified from the sample. The nucleic acids may thenbe tagged in some manner. Tagging may aid the detection of the sequenceof the nucleic acids in a downstream application, such as by making thenucleic acid compatible for use in a particular type of sequencer.

SUMMARY

The application of current technologies for genetic analysis is oftenimpeded by inefficient sample processing techniques. Also, most nucleicacid sample preparation methods have limited uses in that they only candetect one nucleic acid form at a time. For example, most samplepreparation methods require that a sample be divided so that RNA and DNAcan be processed in parallel. Samples containing low quantities ofnucleic acids, or low quality nucleic acids, may thus not havesufficient material to permit detection of both RNA and DNA, resultingin the possible loss of valuable information about the sample.

The present disclosure overcomes these challenges and others. Many ofthe methods, compositions, systems, and kits provided herein enables theconcurrent processing and detection of multiple different types ofnucleic acids, generally without the need of physically separating ordividing a sample. Such concurrent analysis of different nucleic acidforms in a sample permits more efficient detection of genetic material,and for more accurate and useful genetic analyses.

Provided herein are methods, systems, processes, kits, and reagentcompositions useful for carrying out sample preparation processes forthe analysis of different forms of nucleic acids in a sample. Themethods include methods of processing nucleic acids of multiple forms(e.g., single-stranded DNA, double-stranded DNA, single-stranded RNA,and/or double-stranded RNA) within samples to identify the nucleic acidspresent within the sample. The methods, systems, processes, kits, andreagent compositions provided herein can often be practiced or used in asingle reaction mixture, without the need to separate or divide a sampleinto different portions.

In some embodiments, the methods can be applied to samples that compriseboth DNA and RNA fragments of interest, and result in the analysis ofboth of those nucleic acid forms from a single reaction mixture.Further, these methods may identify fragments in accordance with theiroriginating form in the sample, e.g., as DNA or RNA and/or assingle-stranded or double-stranded, such that downstream analysis mayyield both sequence identification and identification of the chemicaland/or structural form of the original nucleic acid in the sample.

In one aspect, provided herein is a method of performing a primerextension reaction on RNA and DNA, comprising: (a) providing a samplecomprising a mixture of single-stranded DNA and single-stranded RNA, (b)attaching a first adapter to the single-stranded DNA, (c) attaching asecond adapter to the single-stranded RNA, (d) annealing a first primerto the first adapter and annealing a second primer to the secondadapter, (e) extending the annealed first primer on the single-strandedDNA to form double-stranded DNA, and/or (f) extending the annealedsecond primer on the single-stranded RNA to form a double-strandedDNA-RNA hybrid. In some cases, the attaching the first adapter to thesingle-stranded DNA comprises ligating the first adapter to thesingle-stranded DNA. In some cases, the attaching of the first adapterto the single-stranded DNA comprises performing a primer extensionreaction. In some cases, the attaching the first adapter to thesingle-stranded rNA comprises ligating the first adapter to thesingle-stranded rNA. In some cases, the attaching of the first adapterto the single-stranded RNA comprises performing a primer extensionreaction.

In some cases, the first adapter is ligated or attached to the 3′ end ofthe single-stranded DNA. In some cases, the second adapter is ligated orattached to the 3′ end of the single-stranded RNA. In some cases, theligating or attaching of said first adapter and said second adapteroccurs concurrently or within a single reaction mixture. In some cases,extending the first primer on the single-stranded DNA to formdouble-stranded DNA occurs prior to the annealing of the second primerto the second adapter ligated to the end (e.g., 3′ end) of thesingle-stranded RNA. In some cases, the extending the first primer onthe single-stranded DNA to form double-stranded DNA occurs at the sametime as the extending the second primer on the single-stranded RNA toform a double-stranded DNA-RNA hybrid. In some cases, the first adapterand the second adapter have different sequences. In some cases, thefirst adapter and the second adapter have the same sequence. In somecases, the first primer and the second primer have different sequences.In some cases, the first primer and the second primer have the samesequence. In some cases, the extending of the first primer can beperformed using a first polymerase that adds at least one firstnon-templated nucleotide to an end (e.g., 3′ end) of a first primerextension strand, thereby generating a first overhang. In some cases,the extending of the second primer is performed using a secondpolymerase that adds at least one second non-templated nucleotide to anend (e.g, 3′ end) of a second primer extension strand, wherein the atleast one second non-templated nucleotide is different from the at leastone first non-templated nucleotide, thereby generating a secondoverhang.

In some cases, the methods further comprise hybridizing a third adapterto the first overhang and a fourth adapter to the second overhang. Insome cases, the method further comprises sequencing the third and fourthadapters and sequences attached to the third and fourth adapters. Insome cases, the method further comprises (i) identifying sequencesassociated with the third adapter as originating from the DNA in theinitial mixture of single-stranded DNA and single-stranded RNA and (ii)identifying sequences associated with the fourth adapter as originatingfrom the RNA in the initial mixture of single-stranded DNA andsingle-stranded RNA.

In one aspect, provide herein is a method of performing an amplificationreaction on a first RNA and a first DNA, comprising: (a) providing asample comprising a mixture of a first DNA and a first RNA, wherein thefirst DNA does not comprise a sequence complementary to the first RNA,(b) tagging an end (e.g, 3′ end) of the first DNA with a first tagwithout using a transposase, (c) tagging a an end (e.g., a 3′ end) ofthe first RNA such that the first RNA comprises a tag that is identicalto the first tag or is not identical to the first tag, (d) performing anamplification or primer extension reaction on the first DNA with apolymerase that is selective for DNA templates, and (e) synthesizing acomplementary cDNA strand from the first RNA with a reversetranscriptase. In some cases, the first DNA is derived from a bacteriumand the first RNA is derived from a virus. In some cases, the methodfurther comprises sequencing the first DNA and the first RNA.

In one aspect, provide herein is a method of sequencing nucleic acids,comprising: (a) providing a sample comprising a mixture ofdouble-stranded nucleic acids and single-stranded nucleic acids, (b)attaching (e.g., by ligation or primer extension reaction) the firstadapter to the double-stranded nucleic acids (e.g, at the 3′ end of thedouble-stranded nucleic acids), (c) denaturing the double-strandednucleic acids into single-stranded nucleic acids, (d) ligating a secondadapter to the denatured nucleic acids of step c, wherein the secondadapter has a different sequence than the first adapter or has asequence that is identical to that of the first adapter, and/or (e)sequencing the nucleic acids ligated to the first and second adaptersand/or identifying sequences associated with the first adapter as beingdouble-stranded and/or sequences associated with the second adapter asbeing single-stranded.

In some cases, the double-stranded nucleic acids are DNA. In some cases,the double-stranded nucleic acids are RNA. In some cases, thesingle-stranded nucleic acids are RNA. In some cases, thesingle-stranded nucleic acids are DNA. In some cases, the method furthercomprises reducing concatemerization of short sequences. In some cases,the DNA is single-stranded DNA, double-stranded DNA, triple-strandedDNA, or a Holliday junction. In some cases, the RNA is single-strandedRNA, double-stranded RNA, or a ribozyme. In some cases, the DNA iscell-free DNA. In some cases, the RNA is cell-free RNA. In some cases,the sample is selected from the group consisting of blood, plasma,serum, cerebrospinal fluid, synovial fluid, bronchio-alveolar lavage,urine, stool, saliva, nasal swab, and any combination thereof.

In some cases, extending the primer on the single-stranded DNA can beperformed by a DNA polymerase. In some cases, the extending the primeron the single-stranded DNA is performed by Bst 2.0 DNA polymerase. Insome cases, the extending the primer on the single-stranded RNA can beperformed by a polymerase selected from Moloney Murine Leukemia Virus(M-MLV) reverse transcriptase, and a SMARTer reverse transcriptase.

In some cases, a method described herein further comprises sequencingthe amplified products.

In some cases, the ligating the first adapter is performed by a ligaseselected from CircLigase II, Thermostable App-DNA/RNA ligase, T4 RNAligase 1, T4 RNA Ligase 2 truncated, and any combination thereof. Insome cases, the ligating the second adapter is performed using adouble-stranded RNA ligase. In some cases, the ligating the secondadapter is performed using T4 RNA ligase 2 or T4 DNA ligase.

In some cases, a method described herein further comprises adding atleast one non-templated nucleotide to a primer extension strand. In somecases, the at least one non-templated nucleotide is a deoxyadenosine. Insome cases, the at least one non-templated nucleotide is onenon-templated nucleotide. In some cases, the third adapter ligatedcomprises an overhang containing at least one deoxythymidine. In somecases, the method further comprises adding at least one non-templatednucleotide to a primer extension strand of the double-stranded DNA-RNAhybrid. In some cases, the at least one non-templated nucleotide is adeoxycytidine. In some cases, the at least one non-templated nucleotideis added to a 3′ end. In some cases, the at least one non-templatednucleotide is up to eight nucleotides. In some cases, the at least onenon-templated nucleotide is three, four, or five non-templatednucleotides. In some cases, the fourth adapter contains an overhangcomprising at least one deoxyguanosine residue. In some cases, theoverhang comprises at least three deoxyguanosine residues.

In one aspect, provide herein is a method of performing an amplificationreaction on a first RNA and a first DNA, comprising: (a) providing asample comprising a mixture of a first DNA and a first RNA, wherein thefirst DNA is derived from a bacterium and the first RNA is derived froma virus, (b) amplifying the first RNA with a reverse transcriptase thatselectively amplifies RNA, and (c) amplifying the first DNA with apolymerase that selectively amplifies DNA.

In one aspect, provided herein is a method of performing anamplification reaction on a first RNA and a first DNA, comprising: (a)providing a sample comprising a mixture of a first DNA and a first RNA,wherein the first DNA is genomic DNA derived from a first organism andthe first RNA is genomic RNA derived from a second organism, (b)amplifying the first RNA with a reverse transcriptase that selectivelyamplifies RNA, and (c) amplifying the first DNA with a polymerase thatselectively amplifies DNA. In some cases, the first organism can be abacterium and the second organism can be a virus.

Provided herein are methods for concurrent processing of differentnucleic acid forms in a sample. The method can comprise (a) denaturingthe nucleic acid forms in a sample; (b) ligating a first adapter to oneend a first nucleic acid form using a ligase that has a preference for afirst nucleic acid form and ligating a second adapter to one end of asecond nucleic acid form using a ligase that has preference for a secondnucleic acid form; (c) primer extending a first and second ligatednucleic acid forms; (d) ligating a third adapter comprising a primingelement; and (e) amplifying. In some cases, the ligating of the firstadapter to the first nucleic acidf form occurs concurrently with theligating of the second adapter to the second nucleic acid form, or inthe same reaction mixture. In a method disclosed herein, a first nucleicacid form can be a DNA molecule and a second nucleic acid form can beRNA a molecule. In other cases, a first nucleic acid form can be ssDNAand a second nucleic acid form can be ssRNA. A polymerase can comprise aDNA-dependent polymerase and a RT polymerase. The polymerase can beselected from a Bst DNA Polymerase, a Full Length, a Bst DNA Polymerase,a Large Fragment, a Bsu DNA Polymerase, a Crimson Taq DNA Polymerase, aLarge Fragment, Deep VentR™, a DNA Polymerase, a Deep VentR™ (exo-), aDNA Polymerase, a E. coli DNA Polymerase I, a Klenow Fragment (3′→5′exo-), a DNA Polymerase I, a Large (Klenow) Fragment, a LongAmp® Taq DNAPolymerase or Hot Start, a M-MuLV Reverse Transcriptase, a OneTaq® DNAPolymerase or Hot Start, a phi29 DNA Polymerase, a Phusion® Hot StartFlex DNA Polymerase, a Phusion® High-Fidelity DNA Polymerase, a Q5®+Q5®Hot Start DNA Polymerase, a Sulfolobus DNA Polymerase IV, a T4 DNAPolymerase, a T7 DNA Polymerase, a Taq DNA Polymerase, a Therminator™DNA Polymerase, a VentR® DNA Polymerase, a VentR® (exo-) DNA Polymerase,and any combination thereof. In some cases, a RT polymerase can beselected from a WarmStart RTx Reverse Transcriptase, a AMV ReverseTranscriptase, a Superscript IV RT, a M-MLV Rnase H(−), a SMARTerreverse transcriptase, a RevertAid RnaseH(−) RT, a ProtoScript® IIReverse Transcriptase, and any combination thereof. Whereas a ligase canbe selected from a T4 DNA Ligase, a T3 DNA Ligase, a T7 DNA Ligase, a E.coli DNA Ligase, a HiFi Taq DNA Ligase, a 9° N™ DNA Ligase, a Taq DNALigase, a SplintR® Ligase, a Thermostable 5′ AppDNA/RNA Ligase, a T4 RNALigase, a T4 RNA Ligase 2, a T4 RNA Ligase 2 Truncated, a T4 RNA Ligase2 Truncated K227Q, a T4 RNA Ligase 2, a Truncated KQ, a RtcB Ligase, aCircLigase II, a CircLigase ssDNA Ligase, a CircLigase RNA Ligase, aAmpligase® Thermostable DNA Ligase, and any combination thereof. Themethod described herein can further comprise a detecting step, whereinthe detecting can be performed by a real-time PCR, sequencing, a digitaldroplet PCR, or a microarray detection assay. Sequencing can comprise anext generation sequencing, a massively-parallel sequencing, apyrosequencing, a sequencing-by-synthesis, a single molecule real-timesequencing, a polony sequencing, a DNA nanoball sequencing, a heliscopesingle molecule sequencing, a nanopore sequencing, a Sanger sequencing,a shotgun sequencing, or a Gilbert's sequencing assay.

Provided herein are methods for concurrent processing of differentnucleic acid forms in a sample. In some cases, the method can comprise:(a) denaturing a nucleic acid forms in a sample; (b) ligating a firstadaptor to one end of a first nucleic acid form using a first ligasethat has a preference for the first nucleic acid form and ligating asecond adapter to one end of a second nucleic acid form using a secondligase that has a preference for the second nucleic acid form, whereinthe first adapter and the second adapter comprise an identifyingsequence that is different from each other; and (c) detecting theligated nucleic acid forms.

Further provided are reaction mixtures comprising: an adapter; a firstligase that has a preference for a first nucleic acid form; a secondligase that has a preference for a second nucleic acid form; and abuffer. The reaction mixture can further comprise a polymerase and/or aRT polymerase described herein. In some cases, components of thereaction mixtures can be liquid, dry, or a combination thereof.

In other reaction mixtures provided herein, the reaction mixture cancomprise: a ligase; a DNA-dependent polymerase that has non-templatedactivity, wherein the non-templated base can be N1; and a RT polymerasethat has non-templated activity, wherein the non-templated base can beN2, wherein N1 and N2 can be different nucleic acid bases. In oneinstance, the DNA-dependent polymerase can be selected from an A- andB-family DNA polymerases, a KOD XL, KOD (exo-), a Bst 2.0, aTherminator, a Deep Vent (exo-), a Pfu DNA polymerase, and aTaq. In somecases, a reverse transcriptase used in the mixture can be selected fromHIV reverse transcriptase, Moloney murine leukemia virus, SuperScriptII™ (ThermoFisher), and SuperScript III™.

Provided herein are kits comprising: an adapter; a first ligase that hasa preference for a first nucleic acid form; a second ligase that has apreference for a second nucleic acid form; and a buffer. In some cases,a kit can further comprise instructions for use. A kit provided hereincan comprise: a ligase; a DNA-dependent polymerase that hasnon-templated activity, wherein the non-templated base is N1; and a RTpolymerase that has non-templated activity, wherein the non-templatedbase can be N2, wherein N1 and N2 can be different nucleic acid bases.Kits provided herein can further comprise instructions for use. TheDNA-dependent polymerase of a kit described herein can be selected froma A- and B-family DNA polymerases, a KOD XL, KOD (exo-), a Bst 2.0, aTherminator, a Deep Vent (exo-), a Pfu DNA polymerase, and aTaq. Whereasa reverse transcriptase can be selected from HIV reverse transcriptase,Moloney murine leukemia virus, SuperScript II™, and SuperScript III™. Akit provided herein may further comprise a control.

Provide herein are methods of sequencing for different nucleic acidsforms. A method of sequencing can comprise: providing a samplecomprising different nucleic acid forms; denaturing the nucleic acidforms in a sample; ligating a first adapter to one end of a firstnucleic acid form using a ligase that has a preference of the firstnucleic acid form; and ligating a second adapter to one end of a secondnucleic acid form using a ligase that has preference of the secondnucleic acid form, wherein the first and the second adapter comprisedifferent identifying sequences; and sequencing the ligated nucleicacids, thereby identifying the different nucleic acid forms in thesample. The method can further comprise amplification by a polymerase,wherein the polymerase can be a DNA-dependent polymerase and/or an RTpolymerase. In some cases, the sequencing described herein can be by anext generation sequencing, a massively-parallel sequencing, apyrosequencing, a sequencing-by-synthesis, a single molecule real-timesequencing, a polony sequencing, a DNA nanoball sequencing, a heliscopesingle molecule sequencing, an nanopore sequencing, a Sanger sequencing,a shotgun sequencing, or a Gilbert's sequencing assay.

Also provided herein are methods for concurrent processing differentnucleic acid forms in a sample. These methods can comprise: denaturingthe nucleic acid forms in a sample; ligating a first adapter to one enda first nucleic acid form and a second nucleic acid form using a ligase;amplifying using a DNA-dependent polymerase that has non-templatedactivity, wherein the non-templated base can be N1; and amplifying usinga RT polymerase that has non-templated activity, wherein thenon-templated base can be N2, wherein N1 and N2 can be different nucleicacid bases. In some cases, a first nucleic acid form or a second nucleicacid form can be DNA, ssDNA, RNA or ssRNA. In some cases theDNA-dependent polymerase can be selected from A- and B-family DNApolymerases, KOD XL, KOD (exo-), Bst 2.0, Therminator, Deep Vent (exo-),Pfu DNA polymerase, and Taq. Whereas, a reverse transcriptase can beselected from HIV reverse transcriptase, Moloney murine leukemia virus,SuperScript II™, and SuperScript III™.

Also provided herein are a method for processing different nucleic acidforms in a sample comprising: (a) denaturing said different nucleic acidforms in a sample, wherein said different nucleic acid forms comprise afirst nucleic acid form and a second nucleic acid form; (b) attaching afirst adapter to said first nucleic acid form and a second adapter tosaid second nucleic acid form; (c) amplifying said first nucleic acidform using a DNA-dependent polymerase that has non-templated activity,wherein said non-templated activity comprises adding at least one N1nucleotide or a first sequence to amplified products of saidamplification of said first nucleic acid form; and (d) amplifying saidsecond nucleic acid form using a reverse transciptase polymerase thathas non-templated activity, wherein said non-templated activitycomprises adding at least one N2 nucloetide or a second sequence toamplified products of said amplification of said second nucleic acidform, wherein said N1 nucleotide and said N2 nucleotide are differentnucleotides or said first sequence is different from said secondsequence.

In some cases, said first nucleic acid form is a DNA molecule or saidsecond nucleic acid form is RNA a molecule. In some cases, said firstnucleic acid form is ssDNA and said second nucleic acid form is ssRNA.In some cases, said DNA-dependent polymerase is selected from A- andB-family DNA polymerases, KOD XL, KOD (exo-), Bst 2.0, Therminator, DeepVent (exo-), Pfu DNA polymerase, and Taq. In some cases, said reversetranscriptase, is selected from HIV reverse transcriptase, Moloneymurine leukemia virus, SuperScript II, and SuperScript III. In somecases, the method further comprises distinguishing said first nucleicacid form from said second nucleic acid form based on said non-templatedactivity of said reverse transcriptase or based on said non-templatedactivity of said DNA-dependent polymerase. In some cases, the methodfurther comprises distinguishing said first nucleic acid form from saidsecond nucleic acid form based on said N1 or N2 nucleotides or saidfirst or second sequences. In some cases, said attaching of said firstadapter or of said second adapter comprises performing a ligationreaction or primer extenstion reaction. In some cases, the attachingoccurs at the 3′ end of the first nucleic acid form or of the 3′end ofthe second nucleic acid form.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entiretiesto the same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows exemplary approaches for processing DNA and RNA in a sampleby adding adapters to single-stranded nucleic acids.

FIG. 2 depicts exemplary techniques to detect various nucleic acid formsin a sample using polymerases with non-template activity.

FIG. 3 depicts ligation/primer extension approaches using polymeraseshaving non-templated activity to detect various nucleic acid forms in asample.

FIG. 4 depicts exemplary approaches to detect various nucleic acid formsin a sample, including approaches using a second adapter that containsboth double-stranded and single-stranded regions.

FIG. 5 depicts exemplary non-templated approaches to detect variousnucleic acid forms in a sample, including an approach using astrand-displacing polymerase.

FIG. 6 depicts a approaches for detecting cell-free nucleic acids, orother low-quality forms, in a sample.

FIG. 7 depicts exemplary primer extension-non-templated approaches usinga successive mode.

FIG. 8 shows exemplary primer extension-non-templated approaches using aconcurrent mode.

FIG. 9 exemplary approaches for distinguishing different structuralforms of the nucleic acids in a sample.

FIG. 10 shows an electrophoric gel illustrating the efficiency ofdifferent DNA and RNA ligases in a single reaction mixture provided bythe disclosure. Lane A1 of the gel shows the molecular ladder (L); LanesB2 and C2 is the product produced using a CircLigase II. Lanes D2 and E2is the product produced using a thermostable App-DNA/RNA ligase. LanesF2 and G2 is the product produced using a T4 RNA ligase 1.

FIG. 11A and FIG. 11B show bar graphs comparing the recovery of theinput DNA and RNA of the starting sample with the final output DNA andRNA detected after conducting the methods of the disclosure. 11A showsrecovery of DNA and RNA product with a SMARTer Reverse Transcriptase.11B shows recovery of the DNA and RNA product with a Bst 2.0 Polymerase.

FIG. 12 shows an electrophoric gel illustrating nucleic acid productsdetected using the methods of the disclosure.

FIG. 13 depicts a primer extension reaction using various reversetranscriptase enzymes.

FIG. 14 depicts a bar graph comparing the performance of an embodimentof the ligation method with a commercial kit by NuGEN. The white barsindicate the number of nucleic acid products detected by the ligationmethod. The hatched bars indicate the number of nucleic acid productsdetected NuGEN method. The x-axis shows the name of the selectedpathogens for the study.

FIG. 15 depicts a bar graph comparing the performance of an embodimentof the ligation method with a commercial kit by NuGEN. The white barsindicate the number of nucleic acid products detected by the ligationmethod. The hatched bars indicate the number of nucleic acid productsdetected NuGEN method. The x-axis shows the name of the selectedpathogens for the study.

FIG. 16 depicts a plot of the quantity versus fragment length for bothhuman chr21 and pathogen cell-free DNA detected using the methodsprovided herein.

FIG. 17 illustrates the activity of polymerases having non-templateactivity. The non-templated nucleotides are indicated by “NNNNN”, whereN could be any nucleotide and any number of Ns can be used. In thisillustration, the non-templated nucleotides are added to the 3′ end ofthe nascent growing strand.

FIG. 18 depicts the non-template activity of a polymerase. The y-axisshows the number of reads detected and the x-axis shows the number ofnon-templated bases added at the 3′ end by the polymerase.

FIG. 19 depicts a computer control system that is programmed orotherwise configured to implement the methods and systems providedherein.

FIG. 20 depicts splint ligase approaches to detect various nucleic acidforms in a sample.

DETAILED DESCRIPTION

The following passages describe different aspects of the invention ingreater detail. Each aspect, embodiment, or feature of the invention maybe combined with any other aspect, embodiment, or feature the inventionunless clearly indicated to the contrary.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs.

“Detect,” as used herein can refer to quantitative or qualitativedetection, including, without limitation, detection by identifying thepresence, absence, quantity, frequency, concentration, sequence, form,structure, origin, or amount of an analyte.

“Nucleic acid” as used herein, can refer to a polymer of nucleotides andis generally synonymous with the term “polynucleotide.” The nucleotidesmay comprise a deoxyribonucleotide, a ribonucleotide, adeoxyribonucleotide analog, ribonucleotide analog, or any combinationthereof. The term “nucleic acid” may also include nucleic acids withmodified backbones. Nucleic acid can be of any length. Nucleic acid mayperform any function, known or unknown. The following are non-limitingexamples of nucleic acids: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA(rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA),micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, primers,mitochondrial DNA, cell-free nucleic acids, viral nucleic acid,bacterial nucleic acid, and genomic DNA. A nucleic acid may comprise oneor more modified nucleotides, such as methylated nucleotides ormethylated nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A nucleic acid may be further modified afterpolymerization, such as by conjugation with a labeling component. Anucleic acid may be single-stranded, double-stranded or have highernumbers strands (e.g., triple-stranded).

“A”, “an”, and “the”, as used herein, can include plural referentsunless expressly and unequivocally limited to one referent.

As used herein, the term “or” is used to refer to a nonexclusive “or”;as such, “A or B” includes “A but not B,” “B but not A,” and “A and B,”unless otherwise indicated.

“Identifying sequence element” can refer to an index, a code, a barcode,a random sequence, an adaptor, an overhang of non-templated nucleicacids, a tag comprising one or more non-templated nucleotides, a primingsequence, or any combination thereof.

As used throughout the specification herein, the term “about” whenreferring to a number or a numerical range means that the number ornumerical range referred to is an approximation within experimentalvariability (or within statistical experimental error), and the numberor numerical range may vary from, for example, from 1% to 15% of thestated number or numerical range. In examples, the term “about” refersto ±10% of a stated number or value.

The term “denaturing”, as used herein, can refer to a process in whichbiomolecules, such as proteins or nucleic acids, lose their structurerelative to their native state. For example, a double-stranded nucleicacid molecule can be denatured into two single-stranded molecules.Denaturing of a protein molecule can lead to loss of its native 3Dstructure to a different structure.

II. General Overview

The present disclosure is directed to methods, compositions, systems,and kits for use in processing biological samples comprising nucleicacids for analysis. The present disclosure provides advantages fornucleic acid analysis of a sample by providing efficient detection,amplification, or quantification of different chemical or structuralforms within a single reaction mixture. For example, the methods can beused for discriminating between RNA and DNA within a sample ordiscriminating between single-stranded and double stranded DNA or RNAwithin a sample. The methods can also be used for quantification of RNAor DNA. In some cases, the products generated by the methods providedherein are analyzed in a downstream assay such as qPCR, ddPCR,microarray, Sanger sequencing, or high-throughput sequencing.

A particular utility of the disclosed methods is that DNA pathogens(e.g., DNA viruses) can be analyzed at the same time as RNA pathogens(e.g., RNA pathogens) in a single reaction mixture, in addition,molecules with different structural forms (e.g., double-stranded,single-stranded) and folding properties (e.g., secondary structures) canbe analyzed at the same time.

A general overview of some embodiments of the methods provided herein isschematically illustrated in FIG. 1. Generally, the method may beginwith obtaining a sample from a subject, such as a human patient, who hasor is suspected of having or is at risk of having a disease, pathogenicinfection, cancer, fetal abnormality or other disorder. The sample maycontain a mixture 100 of double-stranded (ds) DNA, single-stranded (ss)DNA, dsRNA, and ssRNA, or any combination thereof. As shown in FIG. 1,the nucleic acids in the sample may be subjected to denaturation 110,e.g., through application of heat (e.g., 95° C.), for a sufficient timeperiod to ensure that all or most of the nucleic acids within a sampleare present in single-stranded form. In some cases, the sample undergoesa denaturation process in order to remove secondary or tertiarystructure from nucleic acids in the sample (e.g., ssDNA, ssRNA).

The single-stranded nucleic acids (DNA or RNA) may then be subjected toa first adapter ligation step 120 to append a first adapter 160 to the3′ end of the nucleic acid strand. The ligase may be a ligase capable ofligating to both ssRNA and ssDNA (e.g., CircLigase II) or may be a dualligase system that includes a RNA-specific ligase and a DNA-specificligase. In general, the adapters used in the methods provided herein areDNA molecules with a specific or random sequence. The first adapter maycontain additional functional sequences (e.g., one or more ofamplification and sequencing primers, as well as attachment sequences).The first adapter may be modified (e.g., biotinylated or modified with adifferent capture moiety). Streptavidin beads can be used to capturesample nucleic acids ligated to the first adapter as well as unligatedfirst adapter. An excess amount of beads can be used to ensure thebiotinylated first adapters and the ligated sample nucleic acids arecaptured to the beads. As depicted in FIG. 1, the first adapter may besingle-stranded 160. In some cases, identical first adapters are addedat this step, while in other cases, a mixture of first adapters withdifferent sequences is added at this step. Generally, the adapter isappended by a ligation reaction using one or more ligase enzymes, asdescribed further herein, however, other mechanisms may be used as wellto append the first adapter (e.g., random priming), as described furtherherein.

The appended adapter may be used in a primer extension reaction in orderto create a duplex nucleic acid. Generally, the primer 170 used in suchprimer extension reaction may be a DNA or RNA primer that iscomplementary to the sequence of the first adapter or complementary toone sequence of a mixture of first adapters. In some cases, identicalprimers can be added at this step, while in other cases, a mixture ofprimers with different sequences can be added at this step. The primerextension reaction may be performed by a polymerase 130. In some cases,the polymerase may be able to polymerize both DNA and RNA templates.Such polymerase may be used singly or in combination with a DNA-specificor DNA-selective polymerase and/or a RNA-specific or RNA-selectivepolymerase (e.g., reverse transcriptase or RNA-dependent RNA polymerase(e.g., Phi6 RNA polymerase)). In some cases, a polymerase specific foror selective for RNA can be used in combination with a DNA-specific orDNA-selective polymerase. Combinations of polymerases may be usedsequentially or concurrently. In certain embodiments, polymerasescapable of adding one or more non-templated nucleic acid residues to theend of a nascent strand of the duplex may be used, as described in moredetail in FIG. 2 and FIG. 5. By appending such one or more non-templatednucleic acids residues, such polymerases may mark nucleic acids asoriginating from RNA or DNA in the sample and/or from single- ordouble-stranded nucleic acids in the sample, as described furtherherein. In some cases, a reverse transcriptase (e.g., SMARTer RT) and aDNA polymerase (e.g., Bst 2.0 DNA polymerase) are used together to marknucleic acids with one or more non-templated nucleic acid residues(e.g., one or more A's, one or more C's, one or more G's, or one or moreT's). The RT (e.g., SMARTer RT) may mark nucleic acids as originatingfrom RNA by adding terminal one or more residues (e.g., one or moredeoxycytidine residues) to the nascent strand. The DNA polymerase (e.g.,Bst 2.0 DNA polymerase) may mark the nucleic acids as originating fromDNA by adding one or more terminal residues that differ from the one ormore residues used to mark the nucleic acids as originating from RNA(e.g., one or more deoxyadenine residues) to the nascent strand.

After formation of the duplex, a second adapter sequence 180 may beappended to the duplex 140, e.g., added as a double-stranded adapter tothe end opposite to the end to which the first adapter is appended. Thesecond adapter can be a double-stranded adapter with blunt ends or withat least one overhang nucleic acid residue. In some cases, the secondadapter can comprise up to 10 overhang nucleic acid residues. In someother cases, the overhang residues are uniform (e.g., all C's or allA's). In some cases, the overhang residues may be specific for overhangsdeposited by either the Reverse Transcription (RT) or DNA polymeraseused in the primer extension reaction. Adapters with RT-specificoverhangs may further contain an identifying sequence to mark the DNA asresulting from RNA in the starting sample. Likewise, adapters withDNA-polymerase specific overhangs may further contain an identifyingsequence to mark DNA as originating from DNA in the starting sample.

The second adapter is generally composed of double-stranded DNA, but, insome cases, it may contain both DNA and RNA or be entirely made up ofRNA. The second adapter sequence may be ligated to the double-strandedDNA in the reaction mixture using a DNA ligase such as, T4 DNA ligaseand may be ligated to the RNA/DNA hybrid nucleic acids using a ligasesuch as T4 RNA Ligase 2. The second adapter may include additionalfunctional sequences (e.g., one or more of amplification and sequencingprimers, as well as attachment sequences). In some cases, the secondadapter sequence can differentiate or identify the origin of the nucleicacid (e.g., RNA origin vs DNA origin).

A sequential or concurrent process can be used to ligate second adaptersequences to DNA-RNA hybrids or dsDNA using one or more ligases. Forexample, T4 DNA ligase can ligate to dsDNA and to DNA-RNA hybrids. Toselectively ligate a second adapter sequence to one of dsDNA and DNA-RNAhybrids, sequential addition of the ligases (e.g., performing theligation to DNA-RNA hybrids using a RNA ligase first) can be used orconcurrent addition if the RNA ligase ligation rate on DNA-RNA hybridsis sufficiently higher that DNA ligase is not competitive for ligatingto DNA-RNA hybrids.

In some cases, a sequential or successive process is used. In somecases, two or more types of second adapters (e.g., 2a adapter and 2badapter) are used. The 2a adapter type may include a first codeindicating a first nucleic acid (e.g., RNA) origin. The 2b adapter typemay include a second code indicating a second nucleic acid (e.g., DNA)origin. The 2a type of second adapters can be mixed with the sample, andan RNA ligase such as T4 RNA Ligase 2 (truncated) can be added. Theligation reaction to ligate the 2a adapters to DNA:RNA hybrids can beperformed. The sample can be washed to remove excess unligated 2aadapters to prevent 2a adapters from being ligated to dsDNA templates.If the 2a adapter ligation has ligation runs to completion, the washstep can be skipped if unligated 2a adapters are not present. The 2badapters and a DNA ligase such as T4 DNA ligase can be added to ligatethe 2b adapters to dsDNA. Alternatively, the ligation of the 2b adaptersto the dsDNA can be performed first, followed by a wash step to removeexcess unligated 2b adapters, and the ligation of the 2a adapters to theDNA:RNA hybrids can be performed. In general, the more selective orspecific adapter ligation occurs first. The ligated sequences can beamplified and sequenced. The second adapter codes can be used todistinguish between RNA and DNA origins.

In some cases, a concurrent process can be used. For example, if the RNAligase ligation rate on DNA-RNA hybrids is much higher than the ligationrate of the DNA ligase on DNA-RNA hybrids, the DNA ligase may not becompetitive for the DNA-RNA hybrid template. In this case, the RNAligase may selectively ligate the DNA-RNA hybrid and the DNA ligase mayselectively ligate the dsDNA. In some cases, the 2a and 2b adapters maycontain one or more residues in order to selectively hybridize to one ormore overhang residues deposited by a DNA polymerase (e.g., Bst 2.0 DNApolymerase) on dsDNA or a RT (e.g., SMARTer RT) on DNA-RNA hybrids. Incases where the two or more types of second adapters selectivelyhybridize to their templates, ligases may be added concurrently.

The added second adapter sequences can be recognized by a primer inorder to prime primer extension and amplification of the nucleic acidfragments 150. Generally, the primer used in such amplification reactionis a DNA or RNA primer that is complementary to the sequence of thesecond adapter.

In some cases, ligation of a second adapter is not used in the method.Instead, the second adapter may be introduced during the amplificationstage (e.g., 150, the first PCR cycle). For example, the second adapteritself may behave as a primer that recognizes one or more non-templatednucleic acids residues added to the end of a strand by a polymerase suchas SMARTer RT or Bst 2.0. As such, the second adapter may contain anadapter sequence domain as well as a domain that recognizes the one ormore non-templated nucleic acid residues such as one or more C's (e.g.,C, CC, CCC, CCCC, CCCCC, or CCCCCC) or one or more A's (e.g., A) addedby the polymerase. The adapter then primes replication duringamplification 150, resulting in incorporation of the adapter sequenceinto the resulting amplified DNA molecules.

The nucleic acid products of the methods provided by the presentdisclosure may be detected and/or analyzed by any method known in theart. In some cases, a sequencing assay is performed. In some cases, areal-time PCR reaction is performed. In some cases, a microarray-basedassay is performed. In some cases, a digital PCR assay is performed. Aperson skilled in the art will also recognize when new tools developedcan be applied for the analysis of amplified DNA or RNA molecules.

Analysis of the sequencing results may enable detection of RNA and DNAin the originating sample, without necessarily distinguishing betweenthe two types of nucleic acids. In some cases, however, the analysis isused to trace the identity of the originating nucleic acid (e.g.,double-stranded vs. single-stranded, RNA vs. DNA).

In some cases, the second adapter sequence can comprise a sequencingadapter. In some cases, the second adapter sequence can comprise aprimer binding site recognized by a PCR primer, and the PCR primer canalso contain a sequencing primer (e.g., Illumina P5 amplification primersequence or Illumina P7 amplification primer sequence). In some cases,the primer binding site on the second adapter is near or at the 5′ end.

In general, each enzymatic step (e.g., first ligation, primer extension,second ligation, pre-denaturation ligation, etc.) can be performed usingone of three approaches: a single enzyme, successive enzyme addition, orconcurrent enzyme addition. In some cases, a single enzyme is used inone or more enzymatic steps. In some cases, the single enzyme is notselective between RNA and DNA templates. In some cases, successiveenzyme addition of two or more enzymes is used in one or more enzymaticsteps. In some cases, the first enzyme that is added is selective for afirst nucleic acid (e.g., DNA vs. RNA and/or single-stranded vs.double-stranded). In some cases, the second enzyme that is added iseither selective for a second nucleic acid or not selective. In somecases, concurrent enzyme addition of two or more enzymes is used in oneor more enzymatic steps. In some cases, the two or more enzymes areselective for different nucleic acid forms. In some cases, one enzymehas higher selectivity and also has higher activity, and the secondenzyme is not selective or weakly selective. In some cases, the two ormore enzymes are weakly selective or not selective.

The approaches provided herein are generally superior to nucleic acidanalyses that typically focus on a single chemically and structurallyuniform nucleic acid, e.g., only ssDNA, dsDNA, ssRNA, dsRNA, or mRNA,etc. Analysis of a single form may be easier because the different formsgenerally may have different processing needs (reagents, enzymes,cofactors, etc.). However, the results in any given analysis provideonly a partial readout of the nucleic acids present in a given sample.

III. Samples and Analytes

A. Samples

The disclosed methods, systems, compositions, and kits can be used forthe analysis of a wide range of different sample types. The disclosuremay be particularly useful in the evaluation of samples in which thelevel of nucleic acids are of low quality or quantity, by allowinganalysis of a larger fraction of the nucleic acids present in thatsample, regardless of chemical type or structure.

In some cases, a sample can contain cells, tissue, or a bodily fluid. Insome embodiments, a sample can be a liquid or fluid sample. In somecases, a sample can contain a body fluid such as whole blood, plasma,serum, urine, stool, saliva, lymph, spinal fluid, synovial fluid,bronchoalveolar lavage, nasal swab, respiratory secretions, vaginalfluid, amniotic fluid, semen or menses. In some cases, a sample can bemade up of, in whole or in part, cells and/or tissue. In some cases, asample can be made up of a cell-free sample. In some cases, a sample maycomprise nucleic acids (e.g., DNA, RNA, etc.) extracted or purified froma sample (e.g., a clinical sample).

In analyzing genetic composition of a sample (e.g., tissue, blood,serum, etc.) the sample lysis, processing, and extraction of nucleicacid fraction can require different processing steps, buffer solutions,and enzyme systems for the lysis and isolation of the nucleic acidproduct. The methods for processing such different samples types (e.g.,tissue, blood, serum, etc.) are own in the art.

In some embodiments, the obtained sample is a cell-free sample takenfrom a body fluid such as blood, serum, plasma, lymph, urine, or saliva.The cell-free sample may comprise nucleic acids that originated from adifferent site in the body, such as a site of pathogenic infection. Inthe case of blood, serum, lymph, or plasma, the cell-free sample maycontain “circulating” cell-free nucleic acids that originated at adifferent location. In the case of urine, the cell-free nucleic acidsmay be “traveling” cell-free nucleic acids that traveled to the urinefrom a different site in the body. The cell-free samples can be obtainedby removing cells, cell fragments, or exosomes by a known technique suchas by centrifugation or filtration. Samples herein may be biologicalsamples.

In some cases, a sample can be circulating tumor or fetal nucleic acids.Analysis of serum or blood borne nucleic acids, such as circulatingtumor or fetal nucleic acids, e.g., as described in U.S. Pat. Nos.8,877,442 and 9,353,414, or in pathogen identification through, e.g.,analysis of circulating microbial or viral nucleic acids, e.g., asdescribed in Published U.S. Patent Application No. 2015-0133391 andPublished U.S. Patent Application No. 2017-0016048, the full disclosuresof each is incorporated herein by reference in its entirety for allpurposes.

B. Subjects

A sample can be obtained from any subject (e.g., a human subject, anon-human subject, etc.). The subject can be healthy. In some cases, thesubject is a human patient having, suspected of having, or at risk ofhaving, a disease or infection.

A human subject can be a male or female. In some embodiments, the samplecan be from a human embryo or a human fetus. In some embodiments, thehuman can be an infant, child, teenager, adult, or elderly person. Insome cases, the subject is a female subject who is pregnant, suspectedof being pregnant, or planning to become pregnant.

In some embodiments, the subject is a farm animal, a lab animal, or adomestic pet. In some embodiments, the animal can be an insect, dog, acat, a horse, a cow, a mouse, a rat, a pig, a fish, bird, a chicken, ora monkey.

The subject can be an organism, such as a single-celled ormulti-cellular organism. In some embodiments, the sample may be obtainedfrom a plant, fungi, eubacteria, archeabacteria, protest, or anymulticellular organism. The subject may be cultured cells, which may beprimary cells or cells from an established cell line.

In some embodiments, the subject has a genetic disease or disorder, isaffected by a genetic disease or disorder, or is at risk of having agenetic disease or disorder. A genetic disease or disorder can be linkedto a genetic variation such as mutations, insertions, additions,deletions, translocation, point mutation, trinucleotide repeatdisorders, single nucleotide polymorphisms (SNPs), or a combination ofgenetic variations.

The sample can be from a subject who has a specific disease, condition,or infection, or is suspected of having (or at risk of having) aspecific disease, condition, or infection. For example, the sample canbe from a cancer patient, a patient suspected of having cancer, or apatient at risk of having cancer. In other cases, the sample can be froma patient with an infection, a patient suspected of an infection, or apatient at risk of having an infection.

C. Analytes

The disclosure provides for the concurrent detection and geneticanalysis of various chemical and structural analytes found in abiological sample. Analytes can include various chemical forms of a DNAmolecule as well as various forms of a RNA molecule. Analytes can alsoinclude various forms different structural forms of DNA and RNA found ina sample. In some embodiments, the analytes can be particle free (e.g.,such as cell-free). In some embodiments, the analytes can be intact(e.g., exsomes or encapsulated).

Analytes may be any type of nucleic acid including but not limited to:double-stranded (ds) nucleic acids, single stranded (ss) nucleic acids,DNA, RNA, cDNA, mRNA, cRNA, tRNA, ribosomal RNA, dsDNA, ssDNA, miRNA,siRNA, circulating nucleic acids, circulating cell-free nucleic acids,circulating DNA, circulating RNA, cell-free nucleic acids, cell-freeDNA, cell-free RNA, circulating cell-free DNA, cell-free dsDNA,cell-free ssDNA, circulating cell-free RNA, genomic DNA, exosomes,cell-free pathogen nucleic acids, circulating pathogen nucleic acids,mitochondrial nucleic acids, non-mitochondrial nucleic acids, nuclearDNA, nuclear RNA, chromosomal DNA, circulating tumor DNA, circulatingtumor RNA, circular nucleic acids, circular DNA, circular RNA, circularsingle-stranded DNA, circular double-stranded DNA, plasmids, bacterialnucleic acids, fungal nucleic acids, parasite nucleic acids, viralnucleic acids, cell-free bacterial nucleic acids, cell-free fungalnucleic acids, cell-free parasite nucleic acids, viralparticle-associated nucleic acids, viral-particle free nucleic acids orany combination thereof. Analyte nucleic acids may be nucleic acidsderived from pathogens including but not limited to viruses, bacteria,fungi, parasites and any other microbe, particularly an infectiousmicrobe. In some cases, nucleic acids may be derived directly from thesubject, as opposed to a pathogen.

In some instances, the present disclosure provides for analysis ofsingle-stranded nucleic acids. The single-stranded methods provided bythe present disclosure can be applied for more efficient processing ofshorter nucleic acid fragments. In some cases, the single-strandednucleic acids methods, composition, systems, and kits can be applied forpathogen identification in samples that contain circulating or cell-freenucleic acids or highly degraded or low-quality samples such as ancient,formalin-fixed paraffin-embedded (FFPE) samples, or samples which haveundergone many freeze-thaw cycles.

In some instances, the present disclosure provides for analysis of bothdouble-stranded and single-stranded nucleic acids in a sample. In somecases, the subject may have, or be suspected of having, a pathogenicinfection. In this case, the sample from the host subject comprises thehost DNA and RNA, as well as DNA and RNA from a pathogen which can be inthe chemical or structural form of ssRNA, ssDNA, dsRNA, or dsDNA. Thepresent disclosure provides, in some cases, concurrent detection andquantitative analysis of all the nucleic acid forms in an originalsample regardless of their form at the detection stage.

D. Extraction of Analytes

In the methods provided herein, nucleic acids can be isolated from asample using any methods or approaches known in the art. For example,nucleic acids can be extracted using liquid extraction (e.g., Trizol,DNAzol) techniques. Nucleic acids can also be extracted usingcommercially available kits (e.g., QIAamp Circulating Nucleic Acid Kit,Qiagen DNeasy kit, QIAamp kit, Qiagen Midi kit, QIAprep spin kit).

Nucleic acids can be concentrated or precipitated by known methods,including, by way of example only, centrifugation. Nucleic acids can bebound to a selective membrane (e.g., silica) for the purposes ofpurification. Nucleic acids can also be enriched for fragments of adesired length, e.g., fragments which are less than 1000, 500, 400, 300,200 or 100 base pairs in length. Such an enrichment based on size can beperformed using, e.g., PEG-induced precipitation, an electrophoretic gelor chromatography material (Huber et al. (1993) Nucleic Acids Res.21:1061-6), gel filtration chromatography, or TSKgel (Kato et al. (1984)J. Biochem, 95:83-86), which publications are hereby incorporated byreference in their entireties for all purposes.

A nucleic acid sample can be enriched for target polynucleotides (e.g.target nucleic acids), particularly target nucleic acids associated withcondition, disease, or infection and/or a target tissue type. Targetenrichment can be by any means known in the art. For example, thenucleic acid sample may be enriched by amplifying target sequences usingtarget-specific primers (e.g., primers specific for pathogen nucleicacids). The target amplification can occur in a digital PCR format,using any methods or systems known in the art. The nucleic acid samplemay be enriched by capture of target sequences onto an array immobilizedthereon target-selective oligonucleotides. The nucleic acid sample maybe enriched by hybridizing to target-selective oligonucleotides free insolution or on a solid support. The oligonucleotides may comprise acapture moiety which enables capture by a capture reagent. In someembodiments, the nucleic acid sample is not enriched for targetpolynucleotides, e.g., represents a whole genome.

In some embodiments, nucleic acids can be enriched by a pull-downmethod. In some cases, nucleic acids can be hybridized to complementaryoligonucleotides conjugated to a label such as a biotin tag and using,for example, avidin or streptavidin attached to a solid support),targeted PCR, or other methods. Examples of enrichment techniques thatcan be used include but are not limited to: (a) self-hybridizationtechniques in which the major population in a sample of nucleic acidsself-hybridizes more rapidly than the minor population in the sample;(b) depletion of nucleosome-associated DNA from free DNA; (c) removingand/or isolating DNA of specific length intervals; (d) exosome depletionor enrichment; and (e) strategic capture of regions of interest.

Fragmentation & End Modification

The methods can include fragmenting the nucleic acids. In someapplications, the methods do not include fragmenting the nucleic acids,such as, in application with low quality samples or samples containingshort fragments such as certain samples containing cell-free nucleicacids.

Fragmenting of the nucleic acids may be performed by e.g., mechanicalshearing, passing the sample through a syringe, sonication, heattreatment, or a combination thereof. In some cases, shearing may beperformed by mechanical shearing (e.g. ultrasound, hydrodynamic shearingforces), enzymatic shearing (e.g. endonuclease), thermal fragmentation(e.g. incubation at high temperatures), chemical fragmentation (e.g.alkaline solutions, divalent ions). In some cases, fragmenting can beperformed by using an enzyme, including a nuclease, or a transposase.Nucleases used for fragmenting may comprise restriction endonucleases,homing endonucleases, nicking endonucleases, high fidelity restrictionenzymes, or any enzyme disclosed herein. The methods may comprisefragmenting the target nucleic acids into fragments of certain length,e.g., 10, 25, 50, 60, 80, 100, 120, 140, 160, 200, 500, or 1000 bp orgreater in length.

The lengths of the nucleic acids may vary. The nucleic acids or nucleicacid fragments (e.g., dsDNA fragments, RNA, or randomly sized cDNA) canbe less than 1000 bp, less than 500 bp, less than 200 bp, or less than100 bp. The DNA fragments can be about 40 to about 100 bp, about 50 toabout 125 bp, about 100 to about 200 bp, about 150 to about 400 bp,about 300 to about 500 bp, about 100 to about 500, about 400 to about700 bp, about 500 to about 800 bp, about 700 to about 900 bp, about 800to about 1000 bp, or about 100 to about 1000 bp or more. In some cases,the nucleic acids or nucleic acid fragments (e.g., dsDNA fragments, RNA,or randomly sized cDNA) can be within the range from about 20 to about200 bp, such as within the range from about 40 to about 100 bp.

The ends of dsDNA fragments can be polished (e.g., blunt-ended). Theends of DNA fragments can be polished by treatment with a polymerase.Polishing can involve removal of 3′ overhangs, fill-in of 5′ overhangs,or a combination thereof. The polymerase can be a proofreadingpolymerase (e.g., comprising 3′ to 5′ exonuclease activity). Theproofreading polymerase can be, e.g., a T4 DNA polymerase, Pol 1 Klenowfragment, or Pfu polymerase. Polishing can comprise removal of damagednucleotides (e.g., abasic sites), using any means known in the art.

IV. Denaturation

The methods of the disclosure can include the denaturing of nucleicacids from a sample. The denaturation may cause all or most of thedouble-stranded nucleic acids within the sample to becomesingle-stranded. In some cases, the denaturation removes secondary ortertiary structure from double-stranded or single-stranded nucleicacids. As such, any type of sample may be subjected to the denaturationstep, including samples that contain only double-stranded nucleic acids,only single-stranded nucleic acids, or a mixture of double-stranded andsingle-stranded nucleic acids. In some cases, the single-strandednucleic acids in the sample are there as a result of being subjected todenaturation. In some cases, however, the nucleic acids in the sampleare single-stranded because they were originally single-stranded whenthey were obtained from the subject, e.g., single-stranded viral genomicRNA or single-stranded DNA.

A. Heat

The nucleic acids may be denatured using any method known in the art. Insome cases, the denaturation is accomplished by applying heat to thesample for an amount of time sufficient to denature double-strandednucleic acids or to denature secondary and tertiary structures ofdouble-stranded or single-stranded nucleic acids. In general, the samplemay be denatured by heating at 95° C., or within a range from about 65to about 110° C., such as from about 85 to about 100° C. Similarly, thesample may be heated for any length of time sufficient to effectuate thedenaturation, e.g., from about 10 seconds to about 60 minutes. In somecases, long nucleic acids such as intact dsRNA viruses may requirelonger denaturation times. In general, the denaturation is performed inorder to ensure that all or most of the nucleic acids within a sampleare present in single-stranded form.

In some cases, the denaturation may remove secondary and tertiarystructures in single-stranded DNA and/or RNA molecules. Non-limitingexamples of domains of secondary structure that may be removed duringthe denaturation step in include hairpin loops, bulges, and internalloops and any element contributing to folding of the molecule. In somecases, denaturation may not need to be performed, for example when thesample is known to contain only single-stranded nucleic acids or whenthere is a desire to restrict the ultimate analysis to only thesingle-stranded and not the double-stranded nucleic acids in the sample.

B. Chemical and Mechanical

Depending on the application, chemical or mechanical denaturing can beused (e.g., sonication or the like) with the methods.

Chemical denaturation agents that can be used with the methods of thedisclosure include but are not limited to, alkaline agents (e.g. NaOH),formamide, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO),propylene glycol, or urea.

V. Adapter Attachment

The adapters may be attached to the nucleic acids in a sample at one ormore points during the sample preparation process. In some cases, theadapters may be attached by a ligation reaction or by a primer extensionreaction or a combination of both of these reaction types.

In some cases, the adapters may be attached by the ligation reactionmethod using a ligase enzyme that recognizes a particular nucleic acidform or by a primer extension reaction method using a PCR reaction,where the adapter also acts as a primer for a polymerase which acts on aparticular nucleic acid form.

Depending on the contents of the sample and the goal of the geneticassay, the first and second adapters, or iterations using more than twoadapters, can be attached using various different schemes. In someapplications, the first and second adapters, or successive iterations ofadapters, may be attached to ssDNA, dsDNA, ssRNA, dsRNA, DNA, RNA, orDNA/RNA hybrid molecules, or in any combination. Depending on the typeof nucleic molecule in the sample the adapter attached can be eitherdouble-stranded or single-stranded such that the adapter is compatiblewith the nucleic molecules in the sample. For example, in some cases adouble-stranded adapter is attached to a double-stranded nucleic acid.In some applications, it is desirable to protect the adapter ends, forexample by providing an adapter that is duplexed on one end (ordouble-strande) and single-stranded on the other end.

In some adaptor attachment schemes, the first and second adapters can beboth attached using a ligation reaction. In another case, the first andsecond adapters are both attached using a primer extension reactions. Insome cases, the first adapter can be attached by ligation reaction andthe second adapter is attached by primer extension reaction. In somecases, the first adapter can be attached by primer extension and thesecond adapter can be attached by a ligation reaction.

The primer extension reactions can be carried out by a DNA-dependentpolymerase or a RNA dependent polymerase or a combination thereof. Insome cases, the primer extension reaction can be carried out by a DNA orRNA polymerase having strand displacing activity. In some cases, theprimer extension reaction is carried out by a DNA or RNA polymerase thathas non-templated activity. In some other cases, the primer extensionreaction can be carried out by a DNA or RNA polymerase having stranddisplacing activity and a DNA or RNA polymerase that has non-templatedactivity.

A. Adapter Compositions

The present disclosure also provides adapter compositions. In general,the adapter compositions allow for the detection of different nucleicacid forms in a sample. Depending on the starting sample type, whatnucleic acid(s) are being analyzed, the method, and what detectionsystem is being used, an appropriate adapter can be employed (e.g.,particular functional elements or modifications).

In general, an adapter can comprise a polymerase priming sequence, asequence priming sequence, and one or more identifying sequences (e.g.,such as an index, a barcode, a non-templated overhang, a randomsequence, or a combination thereof). For other applications, an adaptercan comprise a polymerase priming sequence, a sequence priming sequence,and one or more identifying sequences, and a label (e.g., radioactivephosphates, biotin, fluorophores, or enzymes). Labels can be added to anadapter if a purification step or particular detection system is desired(e.g., digital PCR, ddPCR, quantitative PCR, microfluidic device,microarray).

In some applications, the adapter can comprise a polymerase primingsequence, one or more identifying sequences, or a label. In otherapplications, as adapter can comprise a polymerase priming sequence, asequence priming sequence, one or more identifying sequences, or a label(e.g., radioactive phosphates, biotin, fluorophores, or enzymes). Insome applications, the first or second adapter does not comprise alabel.

The adapter may be single-stranded or double-stranded. In some cases,the adapter may be a RNA molecule, a DNA molecule, or contain both DNAand RNA (e.g., DNA/RNA hybrid). In some cases, a double-stranded adaptermay be blunt-ended. In other cases, a double-stranded adapter maycontain nucleic acid residue overhang. Such nucleic acid residueoverhangs (or tails) may be used to mark a molecule as originating fromDNA or RNA in the starting sample (e.g. FIG. 1, 100), particularly whenthe overhangs are complementary to an overhang sequence deposited by aRT (e.g., SMARTer RT) and/or a DNA polymerase (e.g., Bst 2.0 DNApolymerase). For example, the adapter overhang may contain one or more Tresidues in order to hybridize to one or more overhang residuesdeposited by a DNA polymerase (e.g., Bst 2.0 DNA polymerase or thelike). Similarly, the adapter overhang may contain one or more Gresidues in order to hybridize to one or more overhang residuesdeposited by a RT (e.g., SMARTer RT or the like).

In some applications, the first adapter can be single-stranded or secondadapter can be double-stranded. In some applications, the first adaptercan be double stranded, or second adapter can be double stranded. Insome applications, the first and second adapter may contain additionalfunctional sequences (e.g., one or more of amplification and sequencingprimers, as well as attachment sequences). In some cases, the adaptersequence contains a barcode or index to indicate whether a nucleic acidderives from a RNA or DNA in the starting sample.

The disclosure also provides for various modifications at the ends ofthe adapters of the present disclosure for better functionality orcompatibility with a particular method and/or assay.

Adapters with Amino Modification

The disclosure provides adapters with amino modifiers (e.g., 3AmMO,/5AmMC6/ or /5AmMC12/). A primary amino group can be attached to anoligonucleotide. Amino modifiers can be positioned at the 5′-end witheither a standard (C6) or longer (C12) spacer arm. Amino modificationscan be positioned at the 3′-end. An example of such as adapter is SEQ IDNO:14, a splinted ligation adapter:/5Sp9/AA/iSp9/CTTCCGATCTNNNNNN/3AmMO/ combined with SEQ ID NO 15.

Adapters with an Adenylated Oligo Modification

The disclosure provides for adapters with 3′ end blocking bydideoxycytosine (ddC). ddC is a dideoxyribonucleoside, a syntheticanalog of deoxycytosine. In ddC, both the 2′- and 3′-positions of theribose have a hydrogen (—H) group substituted for the —OH group, whereasin dC, only the 2′-position is so substituted.

The ddC modification can be used to block the 3′-end of 5′-adenylatedoligos. This type of adapter is useful for to prevent unwanted extensionby a polymerase in a PCR reaction or PCR-based assay. In someembodiments, the adapter can be a 3′-Spacer C3 /3SpC3/. In someembodiments, the adapter can be a dideoxycytosine /3ddC/ is used.

An example of a 3′ end blocking adapter is SEQ ID NO: 2CGACGCTCTTC/3ddC/ SEQ ID NO:1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

Adapters with Phosphate Group Modification

The disclosure provides adapters that are modified by one or morephosphate groups (e.g., /5Phos/). An adapter having 5′ phosphorylationcan be used where the oligo is used as a substrate for DNA ligase. Anadapter having 3′ phosphorylation can be used to inhibit degradation bysome 3′-exonucleases and can to block extension by DNA polymerases.

An example of such as adapter is SEQ ID NO: 4 /5Phos/AGATCGGAAG/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/3BioTEG/.

Adapters with *GA*TC*T Modification

The disclosure provides for adapters modified with a *GA*TC*T sequenceat the ends. An example of such as adapter is SEQ ID NO: 5GTGACTGGAGTTCAGACGTGTGCTCTTCC*GA*TC*T. where * indicate the location ofphosphothiodiester bond between the neighboring nucleotides in thesequence.

Adapters with *T*G*T*A Modification

The disclosure provides for adapters modified with a *T*G*T*A sequenceat the ends. An example of such as adapter is SEQ ID NO: 35Phos/GGAAGAGCGTCGTGTAGGGAAAGAG*T*G*T*A. where * indicate the locationof phosphothiodiester bond between the neighboring nucleotides in thesequence.

Non-limiting examples of adapters that can be used with the disclosureare provided herein. In some embodiments, an extension primer can becomposed of a sequence reverse complementary to the entire or part ofthe 3′-end adapter. In some cases, the sequence can have a 3′-end and5′-end hydroxyls, and may be protected against 3′-end exonucleaseactivity of some DNA-dependent polymerases (e.g. Large Klenow Fragment)by chemical modifications (e.g. phosphothiodiester bond). An example ofthe sequence is e.g. SEQ ID NO: 1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT. Anequivalent sequence protected against 3′-exonuclease activity by LargeKlenow Fragment may be SEQ ID NO: 5GTGACTGGAGTTCAGACGTGTGCTCTTCC*GA*TC*T, where * indicate the location ofphosphothiodiester bond between the neighboring nucleotides in thesequence.

In some embodiments, a second adapter (i.e. 5′-end adapter) may becomposed of two oligos that are full or partial reverse complements ofeach other. The oligos that is actively ligated to the nucleic acidtemplate has 5′-end phosphate, and is protected against degradation byphosphothiodiester bonds at its 3′-end (e.g. SEQ ID NO: 35Phos/GGAAGAGCGTCGTGTAGGGAAAGAG*T*G*T*A). Its hybridizing partner may bepartial or full-length reverse complement with 3′-end deactivatedagainst ligation (e.g. SEQ ID NO: 2 CGACGCTCTTC/3ddC/).

In some embodiments, a single-stranded 3′-end adapter contains aphosphorylated 5′-end with its 3′-end deactivated against ligation. Theoligo can contain a moiety that can be used for immobilization purposes(e.g. biotin, digoxigenin, antigen). An example of such a sequence isSEQ ID NO: 4 /5Phos/AGATCGGAAG/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/3BioTEG/.

In some embodiments, an amplification forward primer for indexing PCRcan contain a 3′-end region that binds the second adapter attached atthe 5′-end side of the original template. The sequence may also containan index region. For example, SEQ ID NO: 6AATGATACGGCGACCACCGAGATCTACACcctgcgaACACTCTTTCCCTACACGACGCTCT T/ whereindex region is indicated with a lower case font.

In some embodiments, an amplification reverse primer for indexing PCRcan contain a 3′-end region that binds the first adapter attached at the3′-end side of the original template. The sequence may also contain anindex region. For example, SEQ ID NO: 10CAAGCAGAAGACGGCATACGAGATatcttgcGTGACTGGAGTTCAGACGTGT where index regionis indicated with a lower case font.

In some embodiments, the first adapter (i.e. the adapter that attachesto the 3′-end of the original template) may be attached by splintligation where a splint oligo is hybridized to e.g. SEQ ID NO: 4. Thenecessary properties of the splint oligos are deactivated 3′- and5′-ends disabling ligation to the splint oligo. In addition, the 3′-endsequence is randomized containing at least 3 random positions. Finally,the 5′-end is fully or partially reverse complementary to the very5′-end of the 3′-end adapter sequence. An example of such sequence maybe ID NO:14 /5 Sp9/AA/iSp9/CTTCCGATCTNNNNNN/3AmMO/. Notation of themodifications adopted from IDT website.

B. Amplification Element

An adapter can comprise an amplification primer that is a primer used tocarry out a polymerase chain reaction (PCR). In some cases, theamplification primer may be a random primer. In some cases, theamplification primer can be a template-specific primer. In other cases,the amplification primer can be complementary to a known non-templatedoverhang known to be added by the polymerase. In some cases, theamplification primer is a P5 primer. In some cases, the amplificationprimer is a P7 primer. In some cases, the amplification primer only partof a P5 or P7 primer. In some cases, depending on the method ofdetection the amplification primer can comprises or more additionalfunctional elements.

C. Identifying Sequence Element

Generally, the identifying sequence elements (e.g., barcode, index, or acombination thereof) comprise a unique sequence. The identifyingsequence element can be added to a particular nucleic acid form by themethods provided herein (e.g., ligation, primer extension or acombination thereof) allowing the identification of each nucleic acidform in a sample. In some embodiments, the identifying sequence elementmay also contain additional functional elements such as primeramplification sites, sequencing priming sites, or sample indexes.

The identifying sequence element or barcodes can be completely scrambled(e.g., randomers of A, C, G, and T for DNA or A, C, G, and U for RNA) orthey can have some regions of shared sequence. For example, a sharedregion on each end may reduce sequence biases in ligation events. Insome cases, a shared region can be about or at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, or 20 common base pairs. Combinations of barcodescan be added to increase diversity. For example, barcodes can be used asidentifiers for well position in a microtiter plate, array, or the like(e.g., 96 different barcodes for a 96-well plate), and another barcodecan be used as an identifier for a plate number (e.g., 24 differentbarcodes for 24 different plates), giving 96×24=2,304 combinations using96+24=120 sequences. Using three or more barcodes per sample can furtherincrease the achievable diversity. In some cases, barcodes may be aboutor at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 200,250, 300, 350, or 400, 500, or 1000 nucleotides (or base pairs) inlength.

Non-Templated Primer Extension to Mark Originating Nucleic Acids

In some embodiments, the identifying sequence elements can benon-templated nucleotides that have been added during a primer extensionreaction using a polymerase that has non-templated activity. Thenon-templated nucleotides can be any nucleotide such as one or more A,G, C, T, or U in any number and in any sequence.

The methods provided herein may include tagging nucleic acids methods(e.g. identification sequences) that allow for subsequent identificationof those sequences deriving or originating from DNA and/or RNA fragmenttemplates in the sample. This is helpful when one wants to determinewhere the nucleic acid originally came from DNA or RNA. In some cases,such tagging occurs during the primer extension step by using a reversetranscriptase (RT) or DNA polymerase that append one or more uniquenon-templated nucleic acid residues to the end, or tail, of the extendednucleic acid strand (FIG. 17). The RT can be any RT that adds one ormore non-templated nucleic acids to the extended nucleic acid strand(e.g., “nascent” strand, cDNA strand). In some cases, the RT is aMoloney Murine Leukemia Virus (MMLV) RT. In some embodiments, the RT isa SMARTer RT enzyme. In particular, a SMARTer RT enzyme typicallyappends anywhere from 1 to 6 non-templated deoxycytidine residues at theterminus of the replicated strand, as shown in FIG. 7 (step 4) and FIG.18, which can serve as a tag or marker of replicated RNA. In some cases,the RT (e.g., SMARTer enzyme) is used together with a DNA polymerasethat adds a different set of non-templated nucleic acid residues to theend of the primer extension product. The DNA polymerase can be any DNApolymerase known to add one or more non-templated nucleic acid residuesto the nascent strand including Bacillus stearothermophilus DNApolymerase I, which owing to a lack of 3′-5′ exonuclease activity, leave3′ overhangs. As shown in FIG. 7 (step 3), the polymerase may be Bst 2.0DNA polymerase, which adds one or more non-templated adenine (A)residues to the nascent strand. However, in some cases, the RT is usedon its own to mark sequences originating from a RNA fragment in theabsence of a marking DNA polymerase.

Similar to the SMARTer RT enzyme, the Bst-polymerase likewise has beenshown to add one or more non-templated adenosine residues to theterminus of the replicated fragment (or nascent strand), again,providing a basis for identifying that which originates from an RNA orDNA template, either by direct detection or by priming with anappropriate tagging sequence. Use of the SMARTer RT enzyme or Bstpolymerase in combination may thus enable differentiation of startingDNA from starting RNA. In some cases, only one polymerase capable ofadding one or more non-templated nucleotides is used in the reaction.For example, SMARTer RT may be used with a DNA polymerase that does notadd non-templated nucleic acids. Adapters recognizing the dC residuesadded by the SMARTer RT may be used in combination with blunt adaptersthat recognize the DNA originally derived from the starting DNA in thereaction mixture. Conversely, adapters recognizing the dA residues addedby the Bst polymerase may be used in combination with blunt adaptersthat recognize the DNA originally derived from the starting RNA in thereaction mixture.

FIG. 7 provides an exemplary scheme for differentiation of starting RNAfrom starting DNA using successive primer extension with DNA polymeraseand reverse transcriptase. As shown, the nucleic acids in the sample maybe subjected to denaturation, e.g., through application of heat, (step1) to ensure that all nucleic acids are present in single-stranded form.In some cases, denaturation may not need to be performed, for examplewhen the sample is known to contain only single-stranded nucleic acidsor when there is a desire to restrict the ultimate analysis to only thesingle-stranded and not the double-stranded nucleic acids in the sample.

The single-stranded nucleic acids (DNA and/or RNA) may be then subjectedto a first adapter ligation step (step 2), to append a firstsingle-stranded adapters to the 3′ end of the nucleic acid strand. Thefirst adapter may contain additional functional sequences (e.g., one ormore of amplification and sequencing primers, as well as attachmentsequences).

Primers specific for the appended adapters may be used to primereplication of the DNA, using a DNA polymerase (e.g., Bst 2.0 DNApolymerase), to create DNA duplexes (step 3) tagged with one or morenon-templated nucleic acid residues (e.g., one or more dA) at the 3′ endof the extended strand. Primers specific for the appended adapters mayalso be used to prime reverse transcription of the RNA, using a reversetranscriptase (e.g., SMARTer RT), to create cDNA duplexes (step 4)tagged with one or more non-templated nucleic acid residues (e.g., 1-6dC residues).

The DNA polymerase-catalyzed primer extension and the reversetranscriptase-catalyzed primer extension may be performed in any order.For example, the DNA polymerase-catalyzed primer extension may beperformed (e.g., using Bst 2.0 DNA polymerase) before the reversetranscriptase-catalyzed primer extension (e.g., using SMARTer RT).Alternatively, the DNA polymerase-catalyzed primer extension may beperformed after the reverse transcriptase-catalyzed primer extension.

In some embodiments, the primer extension (step 3 above) may beperformed using a DNA polymerase and a reverse transcriptaseconcurrently, as shown in FIG. 8. This approach may be performed byconcurrently using a pair of DNA polymerase (e.g., Bst 2.0 DNApolymerase) and reverse transcriptase (e.g., SMARTer RT) that showspecificity for DNA and RNA templates, respectively (FIG. 8, step 3).

The resulting DNA duplexes and/or the cDNA duplexes from both the abovesuccessive and concurrent methods may comprise non-templated sequences,particularly reflected in one or more overhang non-templated residuesthat were added by the polymerase or reverse transcriptase. Thenon-templated sequences of the DNA duplexes may be different from thetag sequences of the cDNA duplexes. For example, the non-templatedsequences of the DNA duplex may be one or more hanging A's (e.g., A),and the non-templated sequences of the cDNA duplexes may be one or morehanging C's (e.g., C, CC, CCC, CCCC, CCCCC, or CCCCCC).

To the DNA duplexes and the cDNA duplexes from step 3 may be then addedsecond adapters (FIG. 7, step 5), e.g., to the end opposite of the firstappended adapters. In some cases, the second adapters added to the DNAduplexes may be different from the second adapters added to the cDNAduplexes. The second adapters to the DNA duplexes may comprise asequence hybridizing to a sequence of the DNA duplexes (e.g., the tagsequences of the DNA duplexes). The second adapters to the cDNA duplexesmay comprise a sequence hybridizing to a sequence of the cDNA duplexes(e.g., the tag sequences of the cDNA duplexes). For example, the secondadapters to the DNA duplexes may be double-stranded DNA and comprise oneor more hanging T's that hybridize to the one or more hanging A's of thetag sequence of the DNA duplexes. The sequence of such second adaptersmay then be used to identify originating DNA during the later sequencinganalysis. Likewise, the second adapters to the cDNA duplexes may bedouble-stranded DNA and comprise one or more hanging G's (e.g., G, GG,GGG, GGGG, GGGGG, or GGGGGG) that hybridizes to the one or more hangingC's (e.g., C, CC, CCC, CCCC, CCCCC, or CCCCCC) of the tag sequence ofthe cDNA duplexes (or DNA/RNA hybrid nucleic acids). The sequence ofsuch second adapters may be used to identify originating RNA during thesequencing analysis.

The double-stranded adapters may include additional functional sequences(e.g., one or more of amplification and sequencing primers, as well asattachment sequences). The added adapter sequences may then be used toprime amplification of the nucleic acid fragments (step 6).

The amplicons may then be sequenced. The sequence differences betweenthe second adapters designed to be ligated to the DNA duplexes throughhybridization to the one or more non-templated residues in the DNAduplexes and the second adapters designed to be ligated to the cDNAduplexes by hybridization to the one or more non-templated residues inthe RNA/DNA duplexes may be used to distinguish the amplified productsderived from the RNA and DNA in the original sample.

Alternatively, the additional dC residues appended by the SMARTer RT maybe used to prime amplification with a primer that may include, inaddition to optional sequencing primer and attachment sequences, anindex or marker sequence that specifically identifies the cDNA productfrom RNA reverse transcription. With reference to FIG. 7 or 8, thetemplate switch oligonucleotide, may be provided to include thisadditional tagging sequence along with the poly dG primer and optionaladditional sequencing primer (e.g., R1 and R2) and/or attachmentsequences (e.g., p5 and p′7). Likewise, the additional dA residue orresidues appended by the Bst 2.0 DNA polymerase may be used to primeamplification with a primer that may include, in addition to optionalsequencing primer and attachment sequences, an index or marker sequencethat specifically identifies the DNA product from the DNA polymeraseextension reaction. With reference to FIG. 7 or 8, the template switcholigonucleotide, may be provided to include this additional taggingsequence along with the poly dT primer and optional additionalsequencing primer (e.g., R1 and R2) and/or attachment sequences (e.g.,P5 and P7).

D. Label Element

The present disclosure also provides for the adapter with one or morelabels. Labels can be added to an adapter when purification is desiredor for using particular detection.

In some embodiments, purification can be achieved be usingoligonucleotides conjugated to a label such as a biotin tag and using,for example, avidin or streptavidin attached to a solid support forpurification or buffer exchange.

Examples of labels that can be used with the disclosure include but arenot limited to any of those known in the art, such as enzymes,fluorophores, radioisotopes, stable free radicals, lummescers, such aschemilummescers, biolummescers and the like, dyes, pigments, enzymesubstrates and other labels. One skilled in the art will choose a labelthat is compatible with the chosen detection method.

E. Ligation & Ligase Enzymes

In some cases, a first or second adapter may be appended to nucleicacids in a sample using a single ligase enzyme or multiple differentligases. In some cases, the single ligase enzyme has the ability toligate an adapter to both DNA and RNA target molecules. As used herein,the term “pan-ligase” is used to refer to a single ligase with theability to ligate an adapter to both DNA and RNA targets. When multipledifferent ligases are used (e.g., a dual ligase system), the ligases mayeach be specific for a target (e.g., DNA-specific or RNA-specific). Insome cases, a dual ligase system may include DNA-specific, RNA-specific,and/or pan-ligases, in any combination. In some cases, the ligase isspecific for double-stranded nucleic acids (e.g., dsDNA, dsRNA, RNA/DNAduplex). An example of a ligase specific for double-stranded DNA andDNA/RNA hybrids is T4 DNA ligase. In some cases, the ligase is specificfor single-stranded nucleic acids (e.g., ssDNA, ssRNA). An example ofsuch ligase is CircLigase II. In some cases, the ligase is specific forRNA/DNA duplexes. In some cases, the ligase is able to work onsingle-stranded, double-stranded, and/or RNA/DNA nucleic acids in anycombination.

Both DNA or/and RNA ligases that may be used with the disclosure.Examples of ligases that can be used with the disclosure include but arenot limited to, T4 DNA Ligase, T3 DNA Ligase, T7 DNA Ligase, E. coli DNALigase, HiFi Taq DNA Ligase, 9° N™ DNA Ligase, Taq DNA Ligase, SplintR®Ligase (also known as. PBCV-1 DNA Ligase or Chlorella virus DNA Ligase),Thermostable 5′ AppDNA/RNA Ligase, T4 RNA Ligase, T4 RNA Ligase 2, T4RNA Ligase 2 Truncated, T4 RNA Ligase 2 Truncated K227Q, T4 RNA Ligase2, Truncated KQ, RtcB Ligase (joins single stranded RNA with a3″-phosphate or 2′,3′-cyclic phosphate to another RNA), CircLigase II,CircLigase ssDNA Ligase, CircLigase RNA Ligase, aor Ampligase®Thermostable DNA Ligase or a combination thereof.

The reaction mixture may include a dual ligase system that uses each ofa DNA ligase and an RNA ligase for carrying out the first ligation step(FIG. 1, 120), to append the first adapter to the nucleic acids in thesample, whether they are DNA or RNA. The DNA ligase in the dual-ligasesystem may preferentially work on DNA over RNA, even in samples thatcontain both RNA and DNA in the same container or tube. Similarly, theRNA ligase may preferentially work on RNA over DNA, even in samples thatcontain RNA and DNA in the same container or tube. In some cases, theligase added at the first ligation step has pan-ligation capabilitiesand is able to ligate the adapter to both the RNA and the DNA strands inthe sample (e.g., CircLigase II). In some cases, a pan-ligase is used incombination with a RNA-specific ligase, a DNA-specific ligase, or with asecond ligase that is also capable of ligating to both RNA and DNA. Incases where more than one ligase is used, the ligases can be addedsimultaneously to the sample. In other cases, the ligases are addedsequentially. In some cases, a single or a dual ligase system may beemployed in order to carry out the second adapter ligation step (FIG. 1,140). In certain embodiments, T4 DNA ligase is used to ligate the secondadapter to the duplex. The enzymes and reaction conditions may beselected to provide sufficient levels of ligation activity of the firstand second adapters to both the DNA and RNA fragments in the sample.

As noted above, a single ligase enzyme may be selected for the reactionsystem that has sufficient ligation activity for each of DNA and RNAsubstrates. In such cases, a ligase may generally be selected that doesnot show any overwhelming preference for either of DNA or RNAsubstrates. Ligases applicable to this system may include, for example,CircLigase II, T4 RNA ligase 1 and 2, including truncated forms, T4 DNAligase, and Thermostable App-DNA/RNA ligases. Of these ligases,CircLigase II may provide less discrimination between DNA or RNAsubstrates, and thus provides an example of a ligase for the firstligation reaction (e.g., FIG. 1, 120). A ligase that can ligate dsDNAand/or DNA-RNA hybrids (e.g., T4 DNA ligase) can be used for the secondligation reaction (e.g., FIG. 1, 140).

Ligases that may be used in the methods provided herein may include, butare not limited to, T4 DNA Ligase, T3 DNA Ligase, T7 DNA Ligase, E. coliDNA Ligase, HiFi Taq DNA Ligase, 9° N™ DNA Ligase, Taq DNA Ligase,SplintR® Ligase, Thermostable 5′ AppDNA/RNA Ligase, T4 RNA Ligase, T4RNA Ligase 2, T4 RNA Ligase 2 Truncated, T4 RNA Ligase 2 TruncatedK227Q, T4 RNA Ligase 2, Truncated KQ, RtcB Ligase, CircLigase II,CircLigase ssDNA Ligase, CircLigase RNA Ligase, Ampligase® ThermostableDNA Ligase, or a combination thereof.

In some cases, the adapters ligated to single-stranded RNA may contain a5′-end modification such as App (e.g., pre-adenylation). The presence ofthe 5′ App modification can enable oligonucleotides to act as directsubstrates for certain ligases and remove the need for ATP. Adapters tosingle-stranded RNA can contain a 5′ adenylation (5′ App) modificationand/or an RNA-identifying code.

Alternatively or additionally, DNA and RNA in a sample can bespecifically marked during the first adapter ligation step and/or duringthe second ligation step. In some cases, a ligase specific for one typeof the nucleic acids is used. For example, a DNA-specific ligase may beused so that adapters are only ligated to the DNA molecules in thesample. In another example, an RNA-specific ligase may be used so thatadapters are only ligated to the RNA molecules in the sample. In certaincases, successive ligation with a first ligase specific to one type ofnucleic acid and a second ligase not discriminating nucleic acids typesare used. For example, successive ligation first with a DNA-specificligase (e.g., CircLigase ssDNA ligase) followed by a ligase that can acton a DNA or RNA template (e.g., CircLigase II) may be used. Sequentialor concurrent first adapter ligation and/or sequential or concurrentsecond adapter ligation may provide the ability to distinguish betweenchemical forms of nucleic acids (e.g., DNA and RNA). The choice ofligation method may depend on the ligase specificities and reactionconditions for each ligase used.

F. Successive Mode of Attachment

The methods provided by the present disclosure can be applied in asuccessive mode, that is more than one enzymatic steps can be applied atseparate steps in the process. In some cases when successive ligation isused, a wash step can be performed between the two ligation reactions toremove the first ligase and excess adapters. For example, successiveligation can be used in the first adapter ligation step (e.g., FIG. 1,120). Biotinylated first adapters with a code for DNA (1a adapters) canbe added to the sample nucleic acids and ligated to ssDNA using a DNAligase. Ligation produces can be immobilized on streptavidin beads.Excess 1a adapters can be washed off. First adapters with a code for RNA(1b adapters) can be added and ligated to ssRNA using an RNA ligase.

In general, for each ligation step (e.g., first ligation, secondligation, pre-denaturation ligation), a single general adapter orspecific adapters can be used. In some cases, a single adapter is addedto all nucleic acids in a ligation step. In some cases, a single adapteris added to a specific group of nucleic acids (e.g., onlysingle-stranded or only double-stranded for a pre-denaturation ligation)in a ligation step. In some cases, different adapters can be added tospecific groups of nucleic acids (e.g., ssDNA, ssRNA, dsDNA, or dsRNA)in a ligation step. In some cases, selectivity can be achieved throughenzymatic selectivity with a wash step in between sequential enzymaticsteps to remove excess unligated adapters. In some cases, selectivitycan be achieved through sequence-specific hybridization to differentoverhangs added by polymerases in the primer extension step.

G. Primer Extension

Adapter Attachment Without Using a Ligase

The first and/or second adapters may be added to the nucleic acids inthe sample with an approach not requiring a ligase-catalyzed reaction.In some cases, the adapters may be added by a primer extension reaction.Such reaction may be performed using random priming with partiallyhybridized oligonucleotides. The oligonucleotide may comprise a primingsequence that hybridizes to the nucleic acids in the sample and anadapter sequence (e.g., a single stranded adapter sequence or adouble-stranded adapter sequence). In some cases, the oligonucleotidesused for random priming contain random sequences. The random sequencescan be optimized to hybridize to a particular genome, such as a humangenome or a pathogen genome. For example, in order to promote priming ofpathogen genomes, the collection of random oligonucleotides (e.g.,13mers) may be partially or entirely depleted of known human sequences.

In some cases, the second adapter may be introduced during theamplification stage (e.g., the first PCR cycle, FIG. 1, 150) without useof a ligase enzyme. For example, the second adapter itself may behave asa primer that recognizes one or more non-templated nucleic acidsresidues added to the end of a nascent strand by a polymerase such asSMARTer RT or Bst 2.0. Such adapter may comprise a domain thathybridizes to the one or more non-templated nucleic acid residues suchas one or more C's (e.g., C, CC, CCC, CCCC, CCCCC, or CCCCCC) or one ormore A's (e.g., A) added by the polymerase. Thus, the second adapter maycomprise one or more G residues in order to hybridize to the one or moreC residues deposited by SMARTer RT. Similarly, the second adapters mayinclude, or may also include, one or more T residues to recognize theone or more A residues added by Bst 2.0 DNA polymerase. The adapter maybe used to prime replication during amplification 150, resulting inincorporation of the adapter sequence into the resulting amplified DNAmolecules. In some cases, the second adapters contain one or moreidentifying sequences to indicate that the original nucleic acid is DNAor RNA. For example, an adapter with one or more T overhang residues mayalso contain a sequence that “marks” the nucleic acid as originatingfrom DNA. An adapter with one or more G overhang residues may alsocontain a sequence that “marks” the nucleic acid as originating fromRNA. Such adapters may also be used in ligation reactions describedabove.

In the first replication or primer extension step (e.g., FIG. 1, 130),the enzymes used in a single reaction mixture may be able to perform theprimer extension reaction against both of a DNA or RNA template with asufficient level of replication of each. Generally, the primer extensionportion of the reaction involves the addition of one or more primers(FIG. 1, 170) that recognize (e.g., hybridize to) the first adaptersattached to the single-stranded DNA and/or RNA. Primer extension alsoinvolves use of a polymerase (e.g., RT, DNA polymerase), dNTPs, andappropriate buffer conditions for the reactions. Following annealing ofthe primer, the polymerases extend the nucleic acid sequence along thelength of the template, thereby forming a nascent nucleic acid (e.g.,DNA) strand. In some cases, the polymerization ends when the end of thetemplate is reached. In other cases, one or more of the polymerases addsone or more non-templated nucleic acids to the end of the nascentstrand, as described further herein. Such non-templated nucleic acidsare at times referred to as “overhangs”, “hanging” nucleotides, or“tails” herein.

Preferably, the primer used in the primer extension reaction is DNA, butin some cases, the primer contains RNA or both RNA and DNA. In somecases, the primer may contain additional sequences in addition to thedomain that is complementary to the adapter sequence. In some cases, theprimer may contain one or more base and/or ribose ring modifications.

In some cases, the polymerase may be able to polymerize both DNA and RNAtemplates. Such polymerase may be used singly or in combination with aDNA-specific polymerase and/or a RNA-specific polymerase. In some cases,a polymerase specific for RNA (e.g., reverse transcriptase) is used incombination with a DNA-specific polymerase. In certain embodiments,polymerases capable of adding one or more non-templated nucleic acidresidues to the end of a nascent strand of the duplex may be used, asdescribed in more detail in FIGS. 7 and 8. Such polymerases may be usedto mark nucleic acids as originating from RNA or DNA in the sampleand/or from single- or double-stranded nucleic acids in the sample, asdescribed further herein.

In some cases, the hybridizing portion of the primer can be 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,or 50 base pairs in length. In some cases, the primer is attached to amultifunctional adapter. The multifunctional adapter can besingle-stranded, double-stranded or both such that the ends areprotected during the reaction.

The enzymes used may include multiple enzymes with differentspecificities or selectivities for DNA or RNA templates in order toachieve sufficient levels of replication of both forms of nucleic acidsin the same reaction mixture, and preferably under the same reactionconditions. By way of example, the reaction mixture may include both DNApolymerases, as well as reverse transcriptases, in order to replicateusing either DNA or RNA as a template with a similar level ofefficiency/replication.

Non-limiting examples of DNA polymerases that can be used in the primerextension step are Bst DNA Polymerase, Full Length, Bst DNA Polymerase,Large Fragment, Bsu DNA Polymerase, Crimson Taq DNA Polymerase, LargeFragment, Deep VentR™ (NEB) DNA Polymerase, Deep VentR™ (exo-) (NEB) DNAPolymerase, E. coli DNA Polymerase I, Klenow Fragment (3′→5′ exo-), DNAPolymerase I, Large (Klenow) Fragment, LongAmp® Taq DNA Polymerase orHot Start (NEB), M-MuLV Reverse Transcriptase, OneTaq® DNA Polymerase orHot Start (NEB), phi29 DNA Polymerase, Phusion® Hot Start Flex DNAPolymerase (NEB), Phusion® High-Fidelity DNA Polymerase (NEB), Q5®+Q5®Hot Start DNA Polymerase (NEB), Sulfolobus DNA Polymerase IV, T4 DNAPolymerase, T7 DNA Polymerase (unmodified), Taq DNA Polymerase, orTherminator™ DNA Polymerase (NEB), VentR® DNA Polymerase (NEB), orVentR® (exo-) DNA Polymerase (NEB), or a combination thereof.

Non-limiting examples of RT polymerases that can be used in the primerextension step are WarmStart RTx Reverse Transcriptase(NEB), AMV ReverseTranscriptase (NEB), Superscript IV RT (Invitrogen), M-MLV Rnase H(−)(Promega), SMARTer reverse transcriptase (Clontech), and RevertAidRnaseH(−) RT (Thermo Scientific), or ProtoScript® II ReverseTranscriptase (NEB), or a combination thereof.

In some applications the primer extension reaction can use a polymerasehaving strand displacing activity. Examples of displacing polymerasethat can be used with the disclosure include but are not limited to,Klenow polymerase, exo-Klenow polymerase, 5′-3′ exo-Klenow polymerase,Bst polymerase, Bst large fragment polymerase, Vent polymerase, Ventpolymerase, Deep Vent (exo-) polymerase, 9° Nm polymerase, Therminatorpolymerase, Therminator II polymerase, MMulV Reverse Transcriptase,phi29 polymerase, or DyNAzyme EXT polymerase, or a combination thereof.

In some cases, a method described herein can comprise successiveaddition of a DNA polymerase followed by a reverse transcriptase orconcurrent addition of a DNA polymerase and a reverse transcriptase. Insome cases, the same primer can be used, or different primers can beused to mark RNA vs. DNA origins. For example, if different primers areused, a first primer that recognizes the adapter ligated in the firstligation and that also contains a DNA code can be added. A DNApolymerase can be used to extend the first primer to form dsDNA. A washstep can remove excess first primer. In some cases, a denaturation stepis added prior to the wash step to selectively denature unextendedprimers that are hybridized to the adapter but to not denature primerextension products (e.g., full length dsDNA). A second primer thatrecognizes the adapter and that contains an RNA code can then be added.A reverse transcriptase can be added to conduct reverse transcription.

Alternatively, a single enzyme system may be employed that hassufficient activity to both template types (e.g., with bothDNA-dependent DNA polymerase activity and RNA-dependent DNA polymeraseactivity) in the single reaction mixture, and preferably under the samereaction conditions. In particular, certain reverse transcriptaseenzymes show lower levels of discrimination between DNA or RNA templatesin replication. For example, as shown in FIGS. 11A and 11B, SMARTerreverse transcriptase polymerases (Clontech) demonstrate the ability tocarry out primer extension/replication against both DNA and RNAtemplates without an excessive preference for single-stranded DNAtemplates (set of 52 bp DNA oligonucleotides) as compared tosingle-stranded RNA templates (50 nt RNA oligonucleotides). As such, incertain cases, the replication step is carried out by incorporating theSMARTer reverse transcriptase in the single reaction mixture to carryout replication/primer extension against both DNA and RNA samplefragment templates in the reaction mixture.

VI. Amplification

The methods of the disclosure can comprise an amplification step using apolymerase chain reaction (PCR). In some applications, there is enoughstarting material in the sample such that no amplification step isnecessarily required.

In some applications, the amplification step of the method performs aforward transcription amplification reaction. In some applications, theamplification step of the method, performs a reverse transcriptionamplification reaction. In some applications the polymerase acts on asingle-stranded nucleic acid molecule. In some applications thepolymerase acts on double-stranded nucleic acid molecule.

In most of the methods that include an amplification step, theamplification step generally serves to amplify the double-stranded DNAresulting from the primer extension reaction. Such dsDNA may contain afirst or second adapter, as described herein. When first and secondadapters are appended to the dsDNA, the amplification may be conductedusing a polymerase chain reaction (PCR) using forward and reverseprimers that, together, recognize the first and second adapter. The PCRreaction maybe conducted with a DNA polymerase. In some cases, the DNApolymerase is identical to the DNA polymerase used during the primerextension step (e.g., Bst 2.0 DNA polymerase). In some cases, DNApolymerase is different from the DNA polymerase used in the primerextension step. Any DNA polymerase known in the art may be used foramplification.

VII. Methods

The methods provided by the disclosure allow for the concurrentdetection of different nucleic acid forms in a sample without therequirement of physical separation or parallel processing. The methodscan be used to distinguishing between DNA and RNA molecules or betweensingle-stranded nucleic acids and double-stranded nucleic acids or acombination thereof. In some embodiments, a method can provide for theconcurrent analysis of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentnucleic acid forms in a sample

A. Ligation Methods

The present disclosure provides ligation methods for the concurrentdetection of different nucleic acid forms within a sample. In someapplications, the method can comprise denaturation, ligation of a firstadapter, primer extension, ligation of a second adapter, andamplification, FIG. 1. In some cases, the ligation method can beconducted concurrently with two different ligation reactions in the samereaction, each with a preference for a different nucleic acid formwithin a sample (e.g., DNA, RNA, dsDNA, dsRNA, ssDNA, ssRNA, etc.).However, in some applications the ligation method can be conducted usingtwo different ligation reactions in successive steps.

In some cases, a ligation method provided herein may involve the use ofone or more ligases that preferentially recognize a particular nucleicacid form (e.g., RNA, DNA, ds nucleic acids, ss nucleic acids, etc.). Insome cases, a ligase that is specific for a certain nucleic acid form(e.g, RNA) may be used with an adapter that is configured to bepreferentially recognized by that ligase. The adapter may contain aknown sequence. For example, a ligase may preferentially ligate theadapter to RNA, thereby “marking” the RNA as RNA. In another example, aligase may preferentially ligate a different adapter with a differentidentifying sequence to DNA, thereby “marking” the DNA as DNA.

Use of ligases that preferentially ligate to a certain form of nucleicacids can generally be used in any of the methods provided herein,particularly during any ligation step. In some cases, two ligases withdifferent nucleic acid preferences are used during the ligation step. Insome cases, one ligaste is used, or more than two ligases. In somecases, the ligases are used in addition to the polymerases withnon-template activity described herein in order to provide additional,or confirmatory information about the identify of a nucleic acid form.

B. Primer-Extension Method

The present disclosure provides primer-extension methods for theconcurrent detection or successive detection of different nucleic acidforms within a sample. In some applications, the method can comprisedenaturing the nucleic acids to be analyzed (e.g., dsDNA, dsRNA, ssDNA,and/or ssRNA), primer extension to add a first adapter, primer extensionto add a second adapter, and/or amplification of the resulting productwith a primer that recognizes the first and/or second adapter. Ingeneral, the primer extension reaction can be carried out with apolymerase and a primer. In some applications, the polymerase can be aDNA-dependent polymerase. In some applications, the polymerase can be aRNA-dependent polymerase.

In some applications, a primer extension method can be conducted using apolymerase that has non-template activity, FIG. 2. In some cases, thepolymerase has a preference for a certain form of template (e.g.,preference for a DNA template over an RNA template, or preference for anRNA template over a DNA template). In some cases, a primer complementaryto the non-templated bases can be used. Such a primer can be used, forexample, to add a second adapter to a developing sequence. In someapplications, the primer extension can be carried out using a polymerasewith strand displacement activity.

C. Ligation-Primer Extension Method

The present disclosure provides a ligation-primer extension method. Theprimer extension method can be particularly useful in applications wheretargeting is desired.

In some embodiments, a ligation-primer extension method can comprise aligation method using a polymerase having non-templated activity todetect various nucleic acid forms in a sample. Such a method cancomprise: attaching a first adapter by ligation, carrying out a primerextension with a polymerase that has non-templated activity to make anoverhang, attaching a second adapter by primer extension, andamplification, FIG. 3. In some embodiments, the second adapter primerextension is performed using an adapter (or primer) that comprises botha sequence of a second adapter and a sequence (e.g., N2N2, as describedherein) that is the reverse complement of the sequence in the overhang.

In some embodiments, a ligation-primer extension method can comprise aligation method using an adapter having both dsDNA and ssDNA regions inorder to detect various nucleic acid forms in a sample. As shown in FIG.4, in some cases, the adapter may have a double-stranded region that canbe ligated to the double-stranded product of a primer extension reaction(e.g., a primer extension reaction that contains the sequence of thefirst adapter). Such adapter may also contain a sequence of a secondadapter, as indicated by the bolded diagonal line in FIG. 4. Such amethod can comprise the following steps: attaching a first adapter byligation, performing a primer extension reaction of the first adapterfollowed by amplification with primers, ligating a second adapter havingboth dsDNA and ssDNA regions, and PCR amplification, FIG. 4. As in manyof the other methods and approaches described herein, the first adaptermay be attached to the nucleic acids by any method, including by aprimer extention reaction using a random primer that recognizes DNAand/or RNA attached to the first adapter or by ligating the firstadapter to a single-stranded nucleic acid, and using a primer thatrecognizes the first adapter to extend the strand.

D. Non-Templated Methods

The present disclosure provides methods involving the use of polymerasescapable of adding non-templated nucleotides to a nucleic acid strand. Ingeneral, a non-templated method provided herein can use one or morepolymerases having non-templated activity. In some cases, the method mayinvolve the use of two polymerases, each with a preference for adifferent template and each that appends a different set ofnon-templated nucleotides to the end of the developing strand. Thenon-templated nucleotides can then be used, downstream, to identify theoriginal form of the nucleic acids. For example, two polymerases, onewith a preference for a DNA template, one with a preference for an RNAtemplate, can be used, wherein each polymerase appends a different setof non-templated nucleotides to the developing strand, thereby “marking”each strand as originating from DNA or RNA. The polymerases may eachhave a preference for any type of nucleic acid form (e.g., DNA, RNA,ssDNA, ssRNA, dsDNA, or dsRNA). In some cases, three or more polymerasesare used, each with a preference for a different nucleic acid form.

In applications where the detection of the different nucleic acids formsin a sample is desired, two polymerases having non-templated activity,should have a different preference in nucleic acid bases for theformation of an overhang, FIGS. 2 and 5. For example, one polymerasewith a preference of a form of nucleic acid (e.g., DNA) can havenon-templated activity for making a “A” overhang, while a secondpolymerase with a preference for a different form of nucleic acid (e.g.,RNA) can have non-templated activity for making a “C” overhang.

In some embodiments, a method using a polymerase with non-templateactivity can be used to distinguish RNA and DNA forms in a samplecomprising dsDNA, ssDNA, dsRNA, ssRNA, in any combination. In somecases, the method may comprise denaturing the nucleic acids in a sampleto produce a sample that contains single-stranded nucleic acids andadding a first adapter to the single-stranded nucleic acids using aprimer extension reaction, where the extension primer contains both thefirst adapter and a 3′-end randomized region that binds to denatured DNAand RNA molecules in the sample. In some cases, the extension primeralso contains a 5′-end region that carries one or more functionalitiesof a first adapter primer as shown in FIG. 1, or as otherwise describedherein. The non-randomized region of the extension primer can beprotected by hybridizing its reverse complement. Once the extensionprimer is hybridized, the primer extension reaction is carried out by apolymerase that can utilize DNA and RNA templates with a known nucleicacid preference for its non-templated activity, such that it introducesan overhang sequence at the 3′-end of the newly synthesized strand is(e.g., “N1,N1” of FIG. 2).

Next, an annealing and amplification step can be carried. Theamplification may be carried out using two amplification primers. Thefirst of these amplification primers may comprise: (1) a primer that isreverse complementary to the known overhang N1 N1 sequence located atthe 3′-end (here, “N2N2”) and (2) a second adapter element positioned atits 5′-end. Generally, the N2N2 primer is attached to the 3′ end of thesecond adapter element in these embodiments. The second of theseamplification primers may recognize the first adapter (that wasinitially added during the first adapter primer extension). Thisamplification step is carried out such that strands that contain bothfirst adapter and second adapter elements may get amplified.

In some cases, the amplification involves use of a polymerase withstrand-displacing activity. As shown in FIG. 2, use of the stranddisplacing polymerase may result in the N1N1N1 . . . strand beingdisplaced with the final product containing the N2N2 . . . sequence andthe sequence of the first adapter. Any strands lacking either a firstadapter or a second adapter elements or both elements will not getamplified (e.g. the original sample DNA and RNA strands) or will getamplified only linearly (e.g. the First Adapter Primer Extension), FIG.2.

In some embodiments, a method using a polymerase with non-templateactivity can comprise: a denaturation step involving denaturing nucleicacids in a sample, a first adapter primer extension step: where thefirst adapter is introduced by primer extension, where the extensionprimer contains a 3′-end randomized region that binds to denatured DNAand RNA molecules. The extension primer may also contain a 5′-end regionthat carries all the functionalities of a first adapter primer as shownin FIG. 1. The non-randomized region of the extension primer can beprotected by hybridizing its reverse complement. Once the extensionprimer is hybridized, the primer extension reaction can, in some cases,be carried out by a SMARTer RT or Bst 2.0 DNA polymerase with a knownnucleic acid preference for its non-templated activity (FIG. 5), suchthat the introduced overhang sequence at the 3′-end of the newlysynthesized strand is known (e.g., for example C,C,C and A,A,A,respectively). Next, an annealing and amplification step can be carried:this step can be carried out using two amplification primers one thathas a primer reverse complementary to the known overhang N1, N1,sequence located at its 3′-end with second adapter elements at its5′-end; (2) a primer containing the same or 5′-end of the first adapter.Next, amplification step is carried out such that strands that containboth first adapter and second adapter elements will get amplified.Finally, a strand displacement step can be carried out using apolymerase with strand-displacing activity: Any strands lacking either afirst adapter or a second adapter elements or both elements will not getamplified (e.g. the original sample DNA and RNA strands) or will getamplified only linearly (e.g. the First Adapter Primer Extension), FIG.2

In general, non-templated activity is the ability of an enzyme (e.g.,DNA polymerases, reverse transcriptases) to synthesize an overhang ofadditional nucleic acid bases in spite of the absence of a template todirect the addition of a particular nucleotide base, FIG. 17. Ingeneral, this can occur at the ends of the template, such as the 3′ or5′ ends of a nucleic acid.

Example DNA polymerases having non-templated activity include but arenot limited to, A- and B-family DNA polymerases, such as (KOD XL, KOD(exo-), Bst 2.0, Therminator, Deep Vent (exo-) Pfu DNA polymerase, orTaq.

Examples of reverse transcriptases having non-templated activity includebut are not limited to, HIV reverse transcriptase, Moloney murineleukemia virus (e.g., SuperScript II™ (ThermoFisher), or SuperScriptIII™ (ThermoFisher).

Non-template activity of an enzyme can be detected using amplificationand sequencing to determine if an enzyme adds nucleotides at the end ofthe template that are non-templated (e.g., overhangs), FIG. 18. Usingthis method, one can determine if the polymerase has non-templateactivity. FIG. 18 shows one embodiment of a RNA polymerase (SMARTer RT)having non-templated activity which can add about one to sixnon-templated nucleotides at the '3 end to form an overhang.

In some applications, the non-templated method can comprise thefollowing steps: denaturation of the sample nucleic acids, attaching afirst adapter by ligation, performing a primer extension reaction usinga primer that recognizes the first adapter using a polymerase that cangenerate non-template nucleotides at the ends (e.g. SMARTer RT, Bst 2.0or the like), attaching the second adapter by primer extension, andamplification.

In some applications, the primer extension step can be carried out usinga polymerase that has non-template activity. In this case anamplification primer would be complementary to the non-templated bases.In some applications, the primer extension can be carried out using apolymerase with strand-displacement activity. In some applications thepolymerase is a DNA-dependent polymerase. In some applications thepolymerase is a RNA-dependent polymerase.

In some embodiments, the non-templated method and be combined with aprimer extension method, referred to as a non-templated-primer extensionmethod. In some embodiments, a non-templated-primer extension method canbe conducted using a successive mode in that the polymerases are usedsuccessively rather than at the same time, FIG. 7. In some embodiments,a non-templated-primer extension method can be using a concurrent mode,involving use of multiple polymerases in the same reaction mixture, FIG.8.

E. Concurrent and Successive Modes

The enzymatic reaction steps of the methods (e.g., ligation, primerextension, and amplification) can be applied successively orconcurrently. FIG. 7 shows some embodiments of a method using asuccessive mode. FIG. 8 shows some embodiments of a method using aconcurrent mode.

Depending on the desired number of nucleic acids to be distinguishedfrom one skilled in the art can using the appropriate number ofidentifying sequences (e.g., such as an index, barcode, non-templatednucleotide overhang, or random sequence).

For example (FIG. 9, step 1) end repair may be performed to generateblunt ends. One can use either the concurrent ligation mode orsuccessive ligation mode to attach an identifying sequence (e.g., anindex, a barcode, a random sequence, a non-templated sequence, orcombination thereof) to the double-stranded nucleic acids in the sample(e.g. dsDNA, and dsRNA), FIG. 9, step 2. To identify dsDNA and dsRNA inthe sample one can use a ligase with preference for double-strandednucleic acids, such that the single-stranded nucleic acids in the sampleare not ligated with an adapter, FIG. 9, step 2. After ligation of theadapter, the double-stranded nucleic acids are “marked” with theadapter. They then can be denatured into single-stranded nucleic acids,that will also contain the tag sequence of the adapter, as shown afterstep 2 in FIG. 9. In some embodiments, one can use a DNA Ligase and RNALigase 2 to attach two different adapters to dsDNA and dsRNA,respectively. Finally, one can proceed with a sample preparation processprovided herein, FIG. 9, step 3, for example proceses as shown in FIG. 7and FIG. 8.

The methods provided by the present disclosure can provide severaladvantages over approaches that use separate, parallel processing toanalyze different nucleic acid forms in a sample. Depending on theapplication, the method may provide one or more advantages such as,decreasing the amount of starting sample required for an analysis, whichin, turn can increase the ability to perform high throughput processeson various types of biological or clinical samples; decrease overallcost, improve the ability to compare the relative abundance or preciseamount of nucleic acids, improve the ability to compare variousstructural forms and chemical structures. In some applications, themethods provided herein can have the advantage of providing moreefficient recovery of short nucleic acid fragments. In someapplications, the methods can have the advantage of decreasing theformation of adapter-dimer during the process.

As will be appreciated, at each of the first ligation step (step 2),first replication step (step 3), and second ligation step (step 4),enzymes and/or reaction conditions would typically be employed andoptimized for the particular form of nucleic acid that is to beanalyzed, e.g., DNA, RNA, or DNA/RNA hybrid. These enzymes and/orreaction conditions may not be optimized or even functional for (or maybe substantially non-functional or lower functioning) toward the otherforms of nucleic acids.

F. Distinguishing Structural Forms Method

The present disclosure provides a for distinguishing between differentstructural forms of nucleic acids. In some applications of the method,an additional adapter ligation step may be performed to help distinguishthe structural forms of nucleic acids in a sample. An adapter may beselectively ligated prior to a denaturation step to only double-stranded(e.g., using a double-stranded nucleic acid ligase such as T4 DNA ligaseand/or T4 RNA ligase 2) or only single-stranded nucleic acids (e.g.,using a single-stranded nucleic acid ligase such as a CircLigaseenzyme). In some cases, one or more adapters can be used, such as anadapter selective for double-stranded nucleic acids, an adapterselective for dsDNA, an adapter selective for dsRNA, an adapterselective for single-stranded nucleic acids, an adapter selective forssDNA, or an adapter selective for ssRNA. For example, double-strandedadapters may be ligated to double-stranded DNA and/or double-strandedRNA in the sample before the denaturation step, as shown in FIG. 9. Thesample may contain a mixture of dsDNA, dsRNA, ssDNA, ssRNA in anycombination.

The sample may undergo an end-repair reaction of the nucleic acids (step1). Concurrent or successive ligation by DNA ligase and RNA ligase 2 maybe conducted in order to attach specific short double-stranded sequences(e.g. adapters) to the double-stranded nucleic acids in the sample,e.g., dsDNA and/or dsRNA (step 2). As a result, the double-strandednucleic acids, but not the single-stranded nucleic acids in the samplecontain the specific short double-stranded sequences (e.g. adapters). Insome cases, an adapter sequence contains a code or index to indicatewhether a nucleic acid derives from a double-stranded or single-strandednucleic acid in the starting sample. The adapters to the double-strandedDNA may comprise different sequences than the adapters to thedouble-stranded RNA. Adapters to double-stranded RNA can contain anRNA-identifying code. A dsRNA ligase can be used to attach the adaptersto dsRNA. Adapters to double-stranded DNA can be designed with aDNA-identifying code. The DNA adapter can be attached to dsDNA using adsDNA ligase.

The adapters may be attached to the double-stranded nucleic acids bysuccessive ligations using DNA ligase and RNA ligase 2, FIG. 9.Alternatively, the adapters may be attached to the double-strandednucleic acids by concurrent ligation by a DNA ligase and an RNA ligasein the same reaction solution. The short sequences in step 2 can bedeactivated to prevent concatemerization. For example, only one 3′ endof these sequences may be left active for ligation, while both 5′-endsand the remaining 3′-end are deactivated by chemical means. Followingthe ligation of the specific short sequences, the sample may bedenatured (step 3) such that it contains entirely single-strandednucleic acids. The steps in FIG. 7 or FIG. 8 may then be conducted inorder to process the RNA and DNA in the sample.

G. Splint Ligase Method

The present disclosure provides a splint ligase method. This processenables discrimination between RNA and DNA by ligating a DNA-specificsequence at the 5′-end of DNA using SplintR Ligase in combination with asplint adapter. In some embodiments, the method can be used todistinguish between the DNA and RNA in a sample. In some embodiments,the method can be used distinguish between the single-stranded anddouble-stranded molecules in a sample.

A splint adapter generally contains a double-stranded region and asingle-stranded region, that may also be described as an overhangregion. In some cases, the overhan region is at the 3′ end of thedouble-stranded region. In some cases, the overhang region is at the 5′end of the double-stranded region. In some cases, the overhang istypically composed of degenerate sites (e.g., N, NN, NNN, NNNN, NNNNN,etc.), usually with a random sequence. The number of degeneratepositions may vary. Generally, a population of splint adapters can beused, wherein the splint adapters have different random N sequences. Thediversity of the sequences may enable hybridization with samplemolecules with a starting unknown identity. In some cases, the NNNNregion has a known sequence, and may be used to hybridize to, forexample, a known nucleic acid, or to an adapter that has been attachedto sample nucleic acid.

In FIG. 20, the depicted splint adapter has a 5′-end overhang ofdegenerate bases. All 5′-ends of this adapter are deactivated againstligation. Similarly the 3′-end that is not indicated in the FIG. 20 isalso deactivated against ligation. Such a splint adapter can hybridizeto 5′-ends of any nucleic acids (i.e. RNA and DNA), but getssuccessfully ligated by SplintR Ligase only to the nucleic acids thathave a 5′-phosphate DNA end.

In general, the method can comprise splint ligation molecule with aSplintR Ligase where the splint ligation molecule is attached to the 5′ends of the DNA and/or RNA within a sample. In some embodiments, thesplint ligation molecules are protected so that only the intended endsare ligatable. The SplintR Ligase preference for the 5′ DNA over the 5′RNA allows heat treatment to detach the splint ligation molecule on onlythe 5′ RNA. Subsequently, a primer extension method can be conducted onboth the DNA ligated to the splint ligation molecule and non-ligatedfree RNA, thereby allowing one to distinguish between the RNA and DNAmolecules in the sample. Non-limiting examples of ligases that can beused are T4 DNA Ligase, T4 RNA Ligase 2, SplintR Ligase, or the like.

In some embodiments, a splint ligase method can comprise the followingsteps: denaturation of the nucleic acids forms in a sample, followed bya first adapter ligation step as shown in (e.g., FIG. 1, Step 120).Next, the 5′-ends of RNA and DNA may be phosphorylated using kinase(e.g., T4 PNK or the like), so that a DNA-specific sequence can be addedto the 5′-end. Next, a splint adapter with 5′-end randomized overhangand SplintR Ligase can be used to ligate DNA-specific sequence to the5′-end of DNA, leaving 5′-end of RNA lacking the DNA-specific sequenceas SplintR Ligase will not process phosphorylated 5′-ends of RNA. After,heat is applied to remove any SplintR Ligase from the RNA. Finally, onecan proceed with the steps shown in FIG. 1 starting with 130 to 150,FIG. 20.

H. Efficiency of Sample Recovery

The methods of the disclosure provide more efficient recovery of theinput staring sample (e.g., before processing). That is, the nucleicacids of the starting sample are recovered in the final sample (e.g.,after processing) at a higher percentage when compared to other nucleicacid sample processing kits, FIG. 14 and FIG. 15.

In some embodiments, the methods of the disclosure provide recovery ofthe starting sample is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percentrecovery of the nucleic acids from the sample compared to the finalprocessed sample.

VIII. Single Reaction Mixture Compositions

The present disclosure provides reaction mixtures. In general, a singlereaction mixture can generate nucleic acid products directed todifferent nucleic acid forms in a sample using a single reactionmixture.

In some applications, the single reaction mixture is provided in asingle liquid or dry format. In other applications, the single reactionmixture is provided in a multiple liquid or dry formats, or acombination thereof.

In some embodiments, a single reaction mixture can require one or morepurification steps. Purification of a single reaction mixture can beaccomplished with the use of one or more labels on the adaptors whichcan be used for purification steps during the single reaction method foroptimal buffer environments for a given enzymes. In some embodiments, asingle reaction mixture of the present disclosure has no purificationsteps.

In some embodiments, a single reaction mixture can comprise an adapter,a ligase that has a preference for a nucleic acid form, and a buffer. Insome embodiments, a single reaction mixture can comprise an adapter, aligase that has a preference for a nucleic acid form, a buffer and aDNA-dependent polymerase.

The ligase in a single reaction mixture can have a preference for aparticular nucleic acid form. A ligase can have a preference for DNAover RNA. A ligase can have a preference for RNA over DNA. A ligase canhave a preference for a single-stranded nucleic acid over adouble-stranded nucleic acid. A ligase can have a preference for adouble-stranded nucleic acid over a single-stranded nucleic acid.

Examples of ligases that can be used in a single reaction mixtureinclude but are not limited to, T4 DNA Ligase, T3 DNA Ligase, T7 DNALigase, E. coli DNA Ligase, HiFi Taq DNA Ligase, 9° N™ DNA Ligase, TaqDNA Ligase, SplintR® Ligase, Thermostable 5′ AppDNA/RNA Ligase, T4 RNALigase, T4 RNA Ligase 2, T4 RNA Ligase 2 Truncated, T4 RNA Ligase 2Truncated K227Q, T4 RNA Ligase 2, Truncated KQ, RtcB Ligase, CircLigaseII, CircLigase ssDNA Ligase, CircLigase RNA Ligase, Ampligase®Thermostable DNA Ligase or a combination thereof.

The polymerase in the single reaction mixture can have a preference orbe dependent on a particular nucleic acid form. For example, polymerasecan be a DNA polymerase or a RT polymerase that is DNA-dependent. Insome other embodiment the polymerase can be a RNA-dependent.

Examples of DNA polymerase that can be used in a single reaction mixtureinclude but are not limited to Bst DNA Polymerase, Full Length, Bst DNAPolymerase, Large Fragment, Bsu DNA Polymerase, Crimson Taq DNAPolymerase, Large Fragment, Deep VentR™ (NEB) DNA Polymerase, DeepVentR™ (exo-) (NEB) DNA Polymerase, E. coli DNA Polymerase I, KlenowFragment (3′→5′ exo-), DNA Polymerase I, Large (Klenow) Fragment,LongAmp® Taq DNA Polymerase or Hot Start (NEB), M-MuLV ReverseTranscriptase, OneTaq® DNA Polymerase or Hot Start (NEB), phi29 DNAPolymerase, Phusion® Hot Start Flex DNA Polymerase (NEB), Phusion®High-Fidelity DNA Polymerase (NEB), Q5®+Q5® Hot Start DNA Polymerase(NEB), Sulfolobus DNA Polymerase IV, T4 DNA Polymerase, T7 DNAPolymerase (unmodified), Taq DNA Polymerase, or Therminator™ DNAPolymerase (NEB), VentR® DNA Polymerase (NEB), or VentR® (exo-) DNAPolymerase (NEB), or a modified form such that its preference for anucleic acid form or its strand displacing activity is increased. Insome embodiments, the single reaction mixture can include a combinationof DNA polymerases.

Non-limiting examples of RT polymerases that can be used in a singlereaction mixture include but are not limited to WarmStart RTx ReverseTranscriptase(NEB), AMV Reverse Transcriptase (NEB), Superscript IV RT(Invitrogen), M-MLV Rnase H(−) (Promega), SMARTer reverse transcriptase(Clontech), and RevertAid RnaseH(−) RT (Thermo Scientific), ProtoScript®II Reverse Transcriptase (NEB), or a modified form such that itspreference for a nucleic acid form or its strand displacing activity isincreased. In some embodiments, the single reaction mixture can includea combination of RT polymerases.

The single desired product regardless of the starting substrate form(e.g., DNA, RNA or DNA/RNA hybrids). While it may be preferred for easethat a single reaction mixture be subject to a single set of reactionconditions to be able to ligate and/or replicate DNA and RNA fragments,it will be appreciated that in some cases, one may alter the bufferconditions applied to the reaction mixture at one or more differentsteps to achieve a desired level of activity in a reaction. For example,depending on the particular reaction to be optimized one may change, forexample, the temperature, divalent co-factors, changing saltconcentration, or addition of one or more additional reagents to a givenreaction mixture at different stages to improve the ligation and/orreplication of one of the forms of nucleic acid in the mixture.

In some embodiments, a reaction mixture can comprise a ligase, aDNA-dependent polymerase that has non-templated activity, wherein thenon-templated base is N1, a RT polymerase that has non-templatedactivity, wherein the non-templated base is N2, wherein N1 and N2 aredifferent nucleic acid bases.

Non-limiting examples of DNA-dependent that can be used in the reactionmixture are: A- and B-family DNA polymerases, KOD XL, KOD (exo-), Bst2.0, Therminator, Deep Vent (exo-), Pfu DNA polymerase, or Taq.

Non-limiting examples of reverse transcriptase that can be used in thereaction mixture are: HIV reverse transcriptase, Moloney murine leukemiavirus, SuperScript II™ (ThermoFisher), or SuperScript III™.

IX. Detection

The methods of the disclosure can include detection of the nucleic acidsforms attached to the adapters provided by the present disclosure. Thedisclosure also provides methods of analysis (e.g., bioinformatics)after detection. Detection can be performed by any means known in theart for nucleic acid detection or future means for nucleic aciddetection. Non-limiting examples of detection means which can be usedare various forms of sequencing, qPCR, ddPCR, microfluidic device, ormicroarray.

A. Sequencing

The methods of the disclosure include the use of a nucleic acidsequencer system (e.g., DNA sequencer, RNA sequencer). The system mayinclude a computer comprising software that performs bioinformaticsanalysis on the sequence information. Bioinformatics analysis caninclude, without limitation, assembling sequence data, detecting andquantifying genetic variants in a sample, including germline variantsand somatic cell variants (e.g., a genetic variation associated withcancer or pre-cancerous condition, a genetic variation associated withinfection).

This disclosure provides methods of analyzing nucleic acids,particularly different forms of nucleic acids present in the samesample. Such analytical methods including sequencing the nucleic acidsas well as bioinformatics analysis of the sequencing results. Thenucleic acids produced according the present methods may be analyzed toobtain various types of information including genomic and RNAexpression. Generally, the analyses provided herein allow forsimultaneous analysis of DNA and RNA in a sample, as well as bothsingle- and double-stranded nucleic acids in a sample. In some cases,the analysis detects both DNA and RNA, yet does not distinguish betweenthe two. In some cases, the analysis detects both DNA and RNA (ordouble- and single-stranded nucleic acids) and also identifies whetherthe originating molecules are DNA, RNA, ssDNA, dsDNA, ssRNA, dsRNA, inany combination. Often, the distinguishing is accomplished by detectingmarkers added to the nucleic acids using adapters described herein.

In some embodiments, the sequencing is performed using a next generationsequencing assay. As used herein, the term “next generation” generallyrefers to any massive, high-throughput sequencing approach including,but not limited to one or more of the following: massively-parallelsignature sequencing, pyrosequencing (e.g., using a Roche 454 sequencingdevice), Illumina (Solexa) sequencing, sequencing by synthesis(Illumina), Ion torrent sequencing, sequencing by ligation (e.g., SOLiDsequencing), single molecule real-time (SNRT) sequencing (e.g., PacificBioscience), polony sequencing, DNA nanoball sequencing, heliscopesingle molecule sequencing (Helicos Biosciences), and nanoporesequencing (e.g., Oxford Nanopore). In some cases, the sequencing assayuses nanopore sequencing. In some cases, the sequencing assay includessome form of Sanger sequencing. In some cases, the sequencing involvesshotgun sequencing, in some cases, the sequencing includes bridge PCR.In some cases, the sequencing is broad spectrum. In some cases, thesequencing is targeted.

In some cases, the sequencing assay comprises a Gilbert's sequencingmethod. In such approach, nucleic acids (e.g., DNA) are chemicallymodified and then cleaved at specific bases. In some cases, a sequencingassay comprises dideoxynucleotide chain termination orSanger-sequencing.

A sequencing-by-synthesis approach may be used in the methods providedherein. In some cases, fluorescently-labeled reversible-terminatornucleotides are introduced to clonally-amplified DNA templatesimmobilized on the surface of a glass flowcell. During each sequencingcycle, a single labeled deoxynucleoside triphosphate (dNTP) may be addedto the nucleic acid chain. The labeled terminator nucleotide may beimaged when added in order to identify the base and may then beenzymatically cleaved to allow incorporation of the next nucleotide.Since all four reversible terminator-bound dNTPs (A, C, T, G) aregenerally present as single, separate molecules, natural competition mayminimize incorporation bias.

In some cases, a method called Single-molecule real-time (SMRT) is used.In such approach, nucleic acids (e.g., DNA) are synthesized in zero-modewave-guides (ZMWs), which are small well-like containers with capturingtools located at the bottom of the well. The sequencing is performedwith use of unmodified polymerase (attached to the ZMW bottom) andfluorescently labelled nucleotides flowing freely in the solution. Thefluorescent label is detached from the nucleotide upon its incorporationinto the DNA strand, leaving an unmodified DNA strand. A detector suchas a camera may then be used to detect the light emissions, and the datamay be analyzed bioinformatically to obtain sequence information.

In some cases, a sequencing by ligation approach is used to sequence thenucleic acids in a sample. One example is the next generation sequencingmethod of SOLiD (Sequencing by Oligonucleotide Ligation and Detection)sequencing (Life Technologies). This next generation technology maygenerate hundreds of millions to billions of small sequence reads at onetime. The sequencing method may comprise preparing a library of DNAfragments from the sample to be sequenced. In some cases, the library isused to prepare clonal bead populations in which only one species offragment is present on the surface of each bead (e.g., magnetic bead).The fragments attached to the magnetic beads may have a universal P1adapter sequence attached so that the starting sequence of everyfragment is both known and identical. In some cases, the method mayfurther involve PCR or emulsion PCR. For example, the emulsion PCR mayinvolve the use of microreactors containing reagents for PCR. Theresulting PCR products attached to the beads may then be covalentlybound to a glass slide. A sequencing assay such as a SOLiD sequencingassay or other sequencing by ligation assay may include a step involvingthe use of primers. Primers may hybridize to the P1 adapter sequence orother sequence within the library template. The method may furtherinvolve introducing four fluorescently labelled di-base probes thatcompete for ligation to the sequencing primer. Specificity of thedi-base probe may be achieved by interrogating every first and secondbase in each ligation reaction. Multiple cycles of ligation, detectionand cleavage may be performed with the number of cycles determining theeventual read length. In some cases, following a series of ligationcycles, the extension product is removed and the template is reset witha primer complementary to the n−1 position for a second round ofligation cycles. Multiple rounds (e.g., 5 rounds) of primer reset may becompleted for each sequence tag. Through the primer reset process, eachbase may be interrogated in two independent ligation reactions by twodifferent primers. For example, the base at read position 5 is assayedby primer number 2 in ligation cycle 2 and by primer number 3 inligation cycle 1.

Sequencing using high-throughput systems may allow detection of asequenced nucleotide immediately after or upon its incorporation into agrowing strand, e.g., detection of sequence in real time orsubstantially real time. In some cases, high throughput sequencinggenerates at least 1,000, at least 5,000, at least 10,000, at least20,000, at least 30,000, at least 40,000, at least 50,000, at least100,000, or at least 500,000 sequence reads per hour. In some cases,each read is at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 120, or at least 150 bases per read. Insome cases, each read is up to 2000, up to 1000, up to 900, up to 800,up to 700, up to 600, up to 500, up to 400, up to 300, up to 200, or upto 100 bases per read. Long read sequencing can include sequencing thatprovides a contiguous sequence read of for example, longer than 500bases, longer than 800 bases, longer than 1000 bases, longer than 1500bases, longer than 2000 bases, longer than 3000 bases, or longer than4500 bases.

In some cases, high-throughput sequencing involves the use of technologyavailable by Illumina's Genome Analyzer IIX, MiSeq personal sequencer,or HiSeq systems, such as those using HiSeq 2500, HiSeq 1500, HiSeq2000, or HiSeq 1000. These machines use reversible terminator-basedsequencing by synthesis chemistry. These machines can do 200 billion DNAor more reads in eight days. Smaller systems may be utilized for runswithin 3, 2, or 1 days or less time. Short synthesis cycles may be usedto minimize the time it takes to obtain sequencing results.

In some cases, high-throughput sequencing involves the use of technologyavailable by ABI Solid System. This genetic analysis platform can enablemassively parallel sequencing of clonally-amplified DNA fragments linkedto beads. The sequencing methodology is based on sequential ligationwith dye-labeled oligonucleotides.

The next generation sequencing can comprise ion semiconductor sequencing(e.g., using technology from Life Technologies (Ion Torrent)). Ionsemiconductor sequencing can take advantage of the fact that when anucleotide is incorporated into a strand of DNA, an ion can be released.To perform ion semiconductor sequencing, a high density array ofmicromachined wells can be formed. Each well can hold a single DNAtemplate. Beneath the well can be an ion sensitive layer, and beneaththe ion sensitive layer can be an ion sensor. When a nucleotide is addedto a DNA, H+ can be released, which can be measured as a change in pH.The H+ ion can be converted to voltage and recorded by the semiconductorsensor. An array chip can be sequentially flooded with one nucleotideafter another. No scanning, light, or cameras can be required. In somecases, an IONPROTON™ Sequencer is used to sequence nucleic acid. In somecases, an IONPGM™ Sequencer is used. The Ion Torrent Personal GenomeMachine (PGM) can do 10 million reads in two hours.

In some cases, high-throughput sequencing involves the use of technologyavailable by Helicos BioSciences Corporation (Cambridge, Mass.) such asthe Single Molecule Sequencing by Synthesis (SMSS) method. SMSS canallow for sequencing the entire human genome in up to 24 hours. SMSS,like the MIP technology, may not require a pre-amplification step priorto hybridization. SMSS may not require any amplification. SMSS isdescribed in part in US Publication Application Nos. 20060024711,20060024678, 20060012793, 20060012784, and 20050100932.

In some cases, high-throughput sequencing involves the use of technologyavailable by 454 Lifesciences, Inc. (Branford, Conn.) such as the PicoTiter Plate device which includes a fiber optic plate that transmitschemiluminescent signal generated by the sequencing reaction to berecorded by a CCD camera in the instrument. This use of fiber optics canallow for the detection of a minimum of 20 million base pairs in 4.5hours.

Methods for using bead amplification followed by fiber optics detectionare described in Marguiles, M., et al. “Genome sequencing inmicrofabricated high-density picolitre reactors”, Nature, doi:10.1038/nature03959, and well as in US Publication Application Nos.20020012930, 20030058629, 20030100102, 20030148344, 20040248161,20050079510, 20050124022, and 20060078909.

In some cases, high-throughput sequencing is performed using ClonalSingle Molecule Array (Solexa, Inc.) or sequencing-by-synthesis (SBS)utilizing reversible terminator chemistry. These technologies aredescribed in part in U.S. Pat. Nos. 6,969,488, 6,897,023, 6,833,246,6,787,308, and US Publication Application Nos. 20040106110, 20030064398,20030022207, and Constans, A., The Scientist 2003, 17(13):36.

In some cases, the next generation sequencing is nanopore sequencing(See e.g., Soni GV and Meller A. (2007) Clin Chem 53: 1996-2001). Ananopore can be a small hole, e.g., on the order of about one nanometerin diameter. Immersion of a nanopore in a conducting fluid andapplication of a potential across it can result in a slight electricalcurrent due to conduction of ions through the nanopore. The amount ofcurrent which flows can be sensitive to the size of the nanopore. As aDNA molecule passes through a nanopore, each nucleotide on the DNAmolecule can obstruct the nanopore to a different degree. Thus, thechange in the current passing through the nanopore as the DNA moleculepasses through the nanopore can represent a reading of the DNA sequence.The nanopore sequencing technology can be from Oxford NanoporeTechnologies, e.g., a GridION system. A single nanopore can be insertedin a polymer membrane across the top of a microwell. Each microwell canhave an electrode for individual sensing. The microwells can befabricated into an array chip, with 100,000 or more microwells (e.g.,more than 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000,900,000, or 1,000,000) per chip. An instrument (or node) can be used toanalyze the chip. Data can be analyzed in real-time. One or moreinstruments can be operated at a time. The nanopore can be a proteinnanopore, e.g., the protein alpha-hemolysin, a heptameric protein pore.The nanopore can be a solid-state nanopore made, e.g., a nanometer sizedhole formed in a synthetic membrane (e.g., SiNx, or SiO2). The nanoporecan be a hybrid pore (e.g., an integration of a protein pore into asolid-state membrane). The nanopore can be a nanopore with an integratedsensors (e.g., tunneling electrode detectors, capacitive detectors, orgraphene based nano-gap or edge state detectors (see e.g., Garaj et al.(2010) Nature vol. 67, doi: 10.1038/nature09379)). A nanopore can befunctionalized for analyzing a specific type of molecule (e.g., DNA,RNA, or protein). Nanopore sequencing can comprise “strand sequencing”in which intact DNA polymers can be passed through a protein nanoporewith sequencing in real time as the DNA translocates the pore. An enzymecan separate strands of a double stranded DNA and feed a strand througha nanopore. The DNA can have a hairpin at one end, and the system canread both strands. In some cases, nanopore sequencing is “exonucleasesequencing” in which individual nucleotides can be cleaved from a DNAstrand by a processive exonuclease, and the nucleotides can be passedthrough a protein nanopore. The nucleotides can transiently bind to amolecule in the pore (e.g., cyclodextran). A characteristic disruptionin current can be used to identify bases.

Nanopore sequencing technology from GENIA can be used. An engineeredprotein pore can be embedded in a lipid bilayer membrane. “ActiveControl” technology can be used to enable efficient nanopore-membraneassembly and control of DNA movement through the channel. In some cases,the nanopore sequencing technology is from NABsys. Genomic DNA can befragmented into strands of average length of about 100 kb. The 100 kbfragments can be made single stranded and subsequently hybridized with a6-mer probe. The genomic fragments with probes can be driven through ananopore, which can create a current-versus-time tracing. The currenttracing can provide the positions of the probes on each genomicfragment. The genomic fragments can be lined up to create a probe mapfor the genome. The process can be done in parallel for a library ofprobes. A genome-length probe map for each probe can be generated.Errors can be fixed with a process termed “moving window Sequencing ByHybridization (mwSBH).” In some cases, the nanopore sequencingtechnology is from IBM/Roche. An electron beam can be used to make ananopore sized opening in a microchip. An electrical field can be usedto pull or thread DNA through the nanopore. A DNA transistor device inthe nanopore can comprise alternating nanometer sized layers of metaland dielectric. Discrete charges in the DNA backbone can get trapped byelectrical fields inside the DNA nanopore. Turning off and on gatevoltages can allow the DNA sequence to be read.

The next generation sequencing can comprise DNA nanoball sequencing (asperformed, e.g., by Complete Genomics, see e.g., Drmanac et al. (2010)Science 327: 78-81). DNA can be isolated, fragmented, and size selected.For example, DNA can be fragmented (e.g., by sonication) to a meanlength of about 500 bp. Adaptors (Ad1) can be attached to the ends ofthe fragments. The adaptors can be used to hybridize to anchors forsequencing reactions. DNA with adaptors bound to each end can be PCRamplified. The adaptor sequences can be modified so that complementarysingle strand ends bind to each other forming circular DNA. The DNA canbe methylated to protect it from cleavage by a type IIS restrictionenzyme used in a subsequent step. An adaptor (e.g., the right adaptor)can have a restriction recognition site, and the restriction recognitionsite can remain non-methylated. The non-methylated restrictionrecognition site in the adaptor can be recognized by a restrictionenzyme (e.g., Acul), and the DNA can be cleaved by Acul 13 bp to theright of the right adaptor to form linear double stranded DNA. A secondround of right and left adaptors (Ad2) can be ligated onto either end ofthe linear DNA, and all DNA with both adapters bound can be PCRamplified (e.g., by PCR). Ad2 sequences can be modified to allow them tobind each other and form circular DNA. The DNA can be methylated, but arestriction enzyme recognition site can remain non-methylated on theleft Ad1 adapter. A restriction enzyme (e.g., Acul) can be applied, andthe DNA can be cleaved 13 bp to the left of the Ad1 to form a linear DNAfragment. A third round of right and left adaptor (Ad3) can be ligatedto the right and left flank of the linear DNA, and the resultingfragment can be PCR amplified. The adaptors can be modified so that theycan bind to each other and form circular DNA. A type III restrictionenzyme (e.g., EcoP15) can be added, EcoP15 can cleave the DNA 26 bp tothe left of Ad3 and 26 bp to the right of Ad2. This cleavage can removea large segment of DNA and linearize the DNA once again. A fourth roundof right and left adaptors (Ad4) can be ligated to the DNA, the DNA canbe amplified (e.g., by PCR), and modified so that they bind each otherand form the completed circular DNA template.

Rolling circle replication (e.g., using Phi 29 DNA polymerase) can beused to amplify small fragments of DNA. The four adaptor sequences cancontain palindromic sequences that can hybridize and a single strand canfold onto itself to form a DNA nanoball (DNB™) which can beapproximately 200-300 nanometers in diameter on average. A DNA nanoballcan be attached (e.g., by adsorption) to a microarray (sequencingflowcell). The flow cell can be a silicon wafer coated with silicondioxide, titanium and hexamehtyldisilazane (HMDS) and a photoresistmaterial. Sequencing can be performed by unchained sequencing byligating fluorescent probes to the DNA. The color of the fluorescence ofan interrogated position can be visualized by a high resolution camera.The identity of nucleotide sequences between adaptor sequences can bedetermined.

B. PCR-Based Detection Methods

Various PCR-based detection methods can be used with the methodsprovided by the present disclosure. Examples of such methods include butare not limited to, sequencing-by-synthesis, digital PCR, ddPCR, orquantitative PCR. In addition, one or all the steps of the methodsprovided by the disclosure can be carried out on a microfluidic device.

C. Microarray

The methods of the present disclosure can be detected by microarray.Microarray maybe desirable targeted applications. In this case, theprobes of the array can be designed to have sequences complementary tosegments the targets of interest and the adapters provided by thepresent disclosure can be labeled with two different fluorophores sothat the microarray apparatus can distinguish between two differentnucleic acid forms.

X. Systems

The methods of the disclosure can include a system. A system can includean apparatus for detection and/or computer control systems withmachine-executable instructions to implement the methods. In someembodiments, the computer control systems are further programmed forconducting genetic analysis.

Detection systems that can be used with the methods of the presentdisclosure can include but are not limited to sequencing, digital PCR,ddPCR, quantitative PCR (e.g. real-time PCR) or by a microfluidicdevice, microarray, or the like.

A. Hardware Systems

Sequencing

A system can include a nucleic acid sequencer (e.g., DNA sequencer, RNAsequencer) for generating DNA or RNA sequence information. The systemmay further include a computer comprising software that performsbioinformatics analysis on the DNA or RNA sequence information.Bioinformatics analysis can include, without limitation, assemblingsequence data, detecting and quantifying genetic variants in a sample,including germline variants and somatic cell variants (e.g., a geneticvariation associated with cancer or pre-cancerous condition, a geneticvariation associated with infection).

Sequencing data may be used to determine genetic sequence information,ploidy states, the identity of one or more genetic variants, as well asa quantitative measure of the variants, including relative and absoluterelative measures. In some cases, sequencing of the genome involveswhole genome sequencing or partial genome sequencing. The sequencing maybe unbiased and may involve sequencing all or substantially all (e.g.,greater than 70%, 80%, 90%) of the nucleic acids in a sample. Sequencingof the genome can be selective, e.g., directed to portions of the genomeof interest. For example, many genes (and mutant forms of these genes)are known to be associated with various cancers. Sequencing of selectgenes, or portions of genes may suffice for the analysis desired.Polynucleotides mapping to specific loci in the genome that are thesubject of interest can be isolated for sequencing by, for example,sequence capture or site-specific amplification.

Digital PCR

In some applications, a system can include an apparatus for digital PCRor droplet based digital PCR. A digital PCR assay can be multiplex, suchthat two or more different analytes or nucleic acid forms are detectedwithin a single partition (e.g. reaction mixture). Amplification of theanalytes can be distinguished by utilizing analyte-specific probeslabeled with different fluorophores or dyes. A digital PCR machine maycomprise a detector the can distinguishably measure the fluorescence ofthe different labels, and thereby detect different analytes.

Measurements can include the determination of copy number, copy numbervariation (e.g., to detect trisomy condition), the status of a singlenucleotide polymorphisms, deletions, duplications, translocations,and/or inversions, which can be the source of disease, susceptibility todisease and/or responsiveness to particular therapeutic treatment.

Real-Time PCR Methodologies

In some applications a system can include an apparatus for real-time PCR(or quantitative PCR (qPCR). A real-time polymerase chain reaction canbe configured for multiplexing by using emission differences of betweentwo or more fluorescent probes or dyes.

Microarray

In some applications, a system can include an apparatus for microarraydetection. Microarray maybe desirable in cases where the methods arebeing applied in a targeted fashion. In some applications, arrays may besubdivided with a gasket into subarrays.

A microarray is device generally contains short single-strandedoligonucleotide probes (e.g., 25- to 70-bp in length) attached to asolid substrate. The probes can be designed to have sequencescomplementary to the targets of interest. Targeted oligos can be addedthe microarray by spotting, spraying, or synthesized in situ through aseries of photocatalyzed reactions.

Microfluidic Devices

In some applications, a system can include a microfluidic apparatus forcarrying put the methods of the disclosure. A microfluidic device usedwith the methods of the disclosure can be configured to perform variousamplification assays including PCR, qPCR, or RT-PCR. In someapplications, the microfluidic device can also can be configured tointegrate pre-PCR or post-PCR assays.

B. Computer Control Systems

The disclosure also provides computer control systems programmed toimplement the methods of the disclosure. FIG. 19 shows a computer system1901 that is programmed or otherwise configured to implement methods ofthe present disclosure.

The computer system 1901 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1905, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1901 also includes memory or memorylocation 1910 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1915 (e.g., hard disk), communicationinterface 1920 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1925, such as cache, othermemory, data storage and/or electronic display adapters. The memory1910, storage unit 1915, interface 1920 and peripheral devices 1925 arein communication with the CPU 1905 through a communication bus (solidlines), such as a motherboard. The storage unit 1915 can be a datastorage unit (or data repository) for storing data. The computer system1901 can be operatively coupled to a computer network (“network”) 1930with the aid of the communication interface 1920. The network 1930 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1930 insome cases is a telecommunication and/or data network. The network 1930can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1930, in some cases withthe aid of the computer system 1901, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1901 tobehave as a client or a server.

The CPU 1905 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1910. The instructionscan be directed to the CPU 1905, which can subsequently program orotherwise configure the CPU 1905 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1905 can includefetch, decode, execute, and writeback.

The CPU 1905 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1901 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1915 can store files, such as drivers, libraries andsaved programs. The storage unit 1915 can store user data, e.g., userpreferences and user programs. The computer system 1901 in some casescan include one or more additional data storage units that are externalto the computer system 1901, such as located on a remote server that isin communication with the computer system 1901 through an intranet orthe Internet.

The computer system 1901 can communicate with one or more remotecomputer systems through the network 1930. For instance, the computersystem 1901 can communicate with a remote computer system of a user.Examples of remote computer systems include personal computers (e.g.,portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® GalaxyTab), telephones, Smart phones (e.g., Apple® iPhone, Android-enableddevice, Blackberry®), or personal digital assistants. The user canaccess the computer system 1901 via the network 1930.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1901, such as, for example, on thememory 1910 or electronic storage unit 1915. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1905. In some cases, thecode can be retrieved from the storage unit 1915 and stored on thememory 1910 for ready access by the processor 1905. In some situations,the electronic storage unit 1915 can be precluded, andmachine-executable instructions are stored on memory 1910.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1901, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1901 can include or be in communication with anelectronic display 1935 that comprises a user interface (UI) 1940 forproviding, an output of a report, which may include a diagnosis of asubject or a therapeutic intervention for the subject. Examples of UI'sinclude, without limitation, a graphical user interface (GUI) andweb-based user interface. The analysis can be provided as a report. Thereport may be provided to a subject, to a health care professional, alab-worker, or other individual.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1905. Thealgorithm can, for example, facilitate the enrichment, sequencing and/ordetection of pathogen or other target nucleic acids.

Information about a patient or subject can be entered into a computersystem, for example, patient background, patient medical history, ormedical scans. The computer system can be used to analyze results from amethod described herein, report results to a patient or doctor, or comeup with a treatment plan.

XI. Applications

The methods, composition, systems, and kits of the disclosure can beused for a variety of applications including personalized medicine,treatment, of any disorders that have a genetic component to drive itspathogenesis or progression. Specifically, the methods of the disclosurecan be applied to a sample to detect, monitor, diagnose, prognose, guidetreatment, or predict the risk of disease.

A. Cancer

The methods of the disclosure can be used for detecting cancer in asubject or for cancer diagnosis. Samples maybe either somatic, germline,or a combination thereof. Samples can be from blood, tissue, or anysample known to harbor the cancer mutation. Cancer cells in the bloodcan be cell-free nucleic acids or as circulating cancer cells, such ascirculating tumor cells (CTCs), cancer stem cells (CSC), hematopoieticstem cells (HSC), and/or endothelial progenitor cells (EPC). The methodscan be used to detect any type circulating cancer cell or cell-freenucleic acids (e.g., DNA or RNA) associated with a tumor.

The methods for cancer can be targeted or non-targeted. In some cases,the methods provided herein can be used to detect specific genes ormutations of interest in the tumor that can be used in the diagnosis ortailoring a cancer treatment for a subject. Such mutations, can includebut are not limited to a mutation associated with cancer progression,drug response, methylation, or a specific cancer gene of interest.

Examples of cancer genes that can be used with the disclosure includebut are not limited to, TP53, CA-125, CEA, PSA, AKT1, ALK, APC, AR,ARAF, ARID1A, ATM, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCNE1, CDH, CDK4,CDK6, CDKN2A, CTNNB1, DDR2, EGFR, ERBB2, ESR1, EZH2, FBXW7, FGFR, FGFR,FGFR3, GATA3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3,KIT, KRAS, MAP2K, MAP2K2, MAPK1, MAPK3, MET, MLH1, MPL, MTOR, MYC, NF1,NFE2L2, NOTCH1, NPM1, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, PTEN, PTPN1,RAF, RB1, RET, RHEB, RHOA, RIT1, ROS1, SMAD4, SMO, STK1, TERT, TSC1, orVHL or any other genes associated with cancer progression.

C. Fetal Health

The methods can be used for detection, diagnosis, or prognosis of fetalhealth (e.g., a IVF embryo or a fetus) in a subject. In some cases, themethods can be used to determine or assess the risk of infection statusof an embryo or fetus. In some cases, the methods can be used for thegenetic assessment for chromosomal aberrations, an inherited conditionincluding but not limited to, autosomal-recessive, dominant, X-linked,or SNP-based genetic conditions in a subject.

The methods for fetal health can be targeted or non-targeted.Non-limiting examples of fetal health conditions that can be used withthe disclosure include, Rh factor, sex of the fetus, Down syndrome(trisomy 21), Trisomy 18, Trisomy 13, Trisomy 16, Trisomy 22, Sexchromosome aneuploidy, or certain genetic disorders or inheritedcondition such as, for example, Prader-Willi syndrome and the like.

D. Infection

The methods can be used for detecting a pathogenic infection in asubject. In some applications, the methods may provide a morecomprehensive view of the state and diversity of the infection in asubject. For example, the identification of both RNA and DNA in a samplemay be useful to detect both RNA and DNA type viruses, as well asbacterial or fungal genomic DNA and transcriptomic RNA. Such process mayalso be able to differentiate between latent infection (e.g., whichmight be indicated by the presence of integrated retroviral DNA) versusactive infection (e.g., which might be indicated by the presence ofviral RNA from intact viral particles). Such analyses may includeanalysis of cell-free, circulating nucleic acids, or degraded nucleicacids e.g., for microbial or viral infection identification.

In addition, the approaches provided herein may yield information aboutparticle-protected nucleic acids, e.g., in exosomes or intact pathogens.

In an infected sample, nucleic acid forms within a given sample mayinclude a variety of different structural forms and hybrids of thoseforms, including DNA and RNA, single and double-stranded forms of these,and structured and unstructured forms of these. By way of example, inthe case of pathogen identification, it will be appreciated thatpathogenic organisms may include a variety of chemical and/or structuralforms of nucleic acids that may be used in their identification.

For example, bacterial and fungal pathogens may include both DNA-basedgenomes and RNA-based transcriptomes, which may be used in theiridentification. Likewise, viral pathogens may include DNA-based genomes,including, e.g., dsDNA viruses (˜24% of viruses) such as human herpesvirus 6, ssDNA viruses (˜9% of viruses) such as microphages, and dsDNART viruses (˜3% of viruses) such as the hepatitis B virus, or RNA-basedgenomes, including ssRNA retroviruses (˜6% of viruses) like HIV, dsRNAviruses (˜9% of viruses) like Rotavirus, (˜) ssRNA viruses (˜18% ofviruses) such as the Ebola virus, (+) ssRNA viruses (˜26% of viruses)like the Hepatitis C virus, and ambisense viruses (˜5% of viruses) likethe Lassa virus.

The methods for detection of an infection can be targeted ornon-targeted. Examples of pathogen infections that can be used with themethods of the disclosure include but are not limited to, Nocardiaspecies, Legionella species, Rickettsia species, Actinomyces species,Mycoplasma species, HACEK organisms (including Haemophilusparainfluenzae, Aggregatibacter aphrophilus, Aggregatibacteractinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, andKingella kingae), Streptobacillus moniliformis, Mycobacteriumtuberculosis complex, Mycobacterium avium complex including M. chimaera,Other nontuberculous mycobacteria, Candida species, Candida auris,Penicillium species, Aspergillus species, Fusarium species, Mucorspecies, Rhizopus species, Rhizomucor species, Scedosporium species,Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum,Cryptococcus neoformans and gattii, Pneumocystis jirovecii, Protozoa,Plasmodium species, Toxoplasma gondii, Acanthamoeba castellanii,Balamuthia mandrillaris, Naegleria fowler, CMV, EBV, Adenovirus, BKPolyomavirus, JC Polyomavirus, Torque Teno Viruses, Abiotrophiadefective, Absidia glauca, Acanthamoeba castellanii, Achromobacterdenitrificans, Achromobacter xylosoxidans, Acidaminococcus intestine,Acidovorax citrulli, Acinetobacter baumannii, Acinetobacter bereziniae,Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacterpittii, Acinetobacter radioresistens, Acinetobacter ursingii, Acremoniumchrysogenum, Acremonium furcatum, Actinobacillus ureae, ActinomaduraLatina, Actinomadura madurae, Actinomucor elegans, Actinomycescardiffensis, Actinomyces europaeus, Actinomyces georgiae, Actinomycesgerencseriae, Actinomyces graevenitzii, Actinomyces israelii,Actinomyces massiliensis, Actinomyces meyeri, Actinomyces neuii,Actinomyces odontolyticus, Actinomyces oris, Actinomyces timonensis,Actinomyces turicensis, Actinomyces viscosus, Adeno-associateddependoparvovirus A, Adeno-associated dependoparvovirus B, Aerococcussanguinicola, Aerococcus urinae, Aerococcus viridans, Aeromonas caviae,Aeromonas hydrophila, Aeromonas schubertii, Aeromonas veronii,Aggregatibacter actinomycetemcomitans, Aggregatibacter aphrophilus,Aggregatibacter segnis, Agrobacterium tumefaciens, Alcaligenes faecalis,Alloiococcus otitis, Alloscardovia omnicolens, Alphapapillomavirus 1,Alphapapillomavirus 2, Alphapapillomavirus 3, Alphapapillomavirus 4,Alphapapillomavirus 5, Alphapapillomavirus 6, Alphapapillomavirus 7,Alphapapillomavirus 8, Alphapapillomavirus 9, Alphapapillomavirus 10,Alphapapillomavirus 11, Alphapapillomavirus 14, Alternaria alternate,Alternaria arborescens, Alternaria brassicicola, Anaerobiospirillumsucciniciproducens, Anaerococcus hydrogenalis, Anaerococcuslactolyticus, Anaerococcus prevotii, Anaerococcus tetradius,Anaeroglobus geminatus, Anaplasma phagocytophilum, Angiostrongyluscantonensis, Angiostrongylus costaricensis, Anisakis simplex, Anncaliiaalgerae, Apophysomyces elegans, Apophysomyces trapeziformis,Arcanobacterium bernardiae, Arcanobacterium haemolyticum,Arcanobacterium pyogenes, Arcobacter butzleri, Arcobacter cryaerophilus,Arcobacter skirrowi, Aspergillus awamori, Aspergillus calidoustus,Aspergillus clavatus, Aspergillus fischeri, Aspergillus flavus,Aspergillus fumigatus, Aspergillus kawachii, Aspergillus lentulus,Aspergillus luchuensis, Aspergillus nidulans, Aspergillus niger,Aspergillus nomius, Aspergillus ochraceoroseus, Aspergillus oryzae,Aspergillus parasiticus, Aspergillus rambellii, Aspergillussclerotiorum, Aspergillus sojae, Aspergillus terreus, Aspergillusudagawae, Aspergillus ustus, Aspergillus westerdijkiae, Atopobiumparvulum, Atopobium rimae, Atopobium vaginae, Aureobasidium melanogenum,Aureobasidium namibiae, Aureobasidium pullulans, Aureobasidiumsubglaciale, Babesia divergens, Babesia microti, Bacillus anthracis,Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillussphaericus, Bacillus subtilis, Bacillus thuringiensis, Bacteroidescaccae, Bacteroides distasonis, Bacteroides eggerthii, Bacteroidesforsythus, Bacteroides fragilis, Bacteroides merdae, Bacteroides ovatus,Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroidesuniformis, Bacteroides vulgatus, Balamuthia mandrillaris, Bartonellaalsatica, Bartonella bacilliformis, Bartonella birtlesii, Bartonellabovis, Bartonella clarridgeiae, Bartonella doshiae, Bartonellaelizabethae, Bartonella grahamii, Bartonella henselae, Bartonellakoehlerae, Bartonella quintana, Bartonella rattaustraliani, Bartonellarochalimae, Bartonella schoenbuchensis, Bartonella taylorii, Bartonellatribocorum, Bartonella vinsonii, Basidiobolus meristosporus, Beauveriabassiana, Beauveria rudraprayagi, Bergeyella zoohelcum,Betapapillomavirus 1, Betapapillomavirus 2, Betapapillomavirus 3,Betapapillomavirus 4, Betapapillomavirus 5, Bifidobacteriumadolescentis, Bifidobacterium breve, Bifidobacterium dentium,Bifidobacterium longum, Bifidobacterium scardovii, Bipolarispapendorfii, BK polyomavirus, Blastocystis hominis, Blastomycesdermatitidis, Bordetella bronchiseptica, Bordetella hinzii, Bordetellaholmesii, Bordetella parapertussis, Bordetella pertussis, Bordetellapetrii, Borrelia burgdorferi, Borrelia crocidurae, Borrelia duttonii,Borrelia hermsii, Borrelia hispanica, Borrelia miyamotoi, Borreliaparkeri, Borrelia persica, Borrelia recurrentis, Borrelia turicatae,Borreliella afzelii, Borreliella garinii, Brevibacillus brevis,Brevibacillus laterosporus, Brevibacterium casei, Brevundimonasdiminuta, Brevundimonas vesicularis, Brucella abortus, Brucella canis,Brucella melitensis, Brucella suis, Brugia malayi, Burkholderiaambifaria, Burkholderia anthina, Burkholderia cenocepacia, Burkholderiacepacia, Burkholderia gladioli, Burkholderia mallei, Burkholderiamultivorans, Burkholderia pseudomallei, Burkholderia pyrrocinia,Burkholderia stabilis, Byssochlamys spectabilis, Campylobacter coli,Campylobacter concisus, Campylobacter corcagiensis, Campylobactercuniculorum, Campylobacter curvus, Campylobacter fetus, Campylobactergracilis, Campylobacter hominis, Campylobacter hyointestinalis,Campylobacter iguaniorum, Campylobacter jejuni, Campylobacter lari,Campylobacter mucosalis, Campylobacter showae, Campylobacter sp. MIT97-5078, Campylobacter sputorum, Campylobacter upsaliensis,Campylobacter ureolyticus, Candida albicans, Candida auris, Candidaboidinii, Candida bracarensis, Candida carpophila, Candida castellii,Candida dubliniensis, Candida ethanolica, Candida famata, Candidaglabrata, Candida intermedia, Candida kefyr, Candida krusei, Candidalusitaniae, Candida nivariensis, Candida orthopsilosis, Candidaparapsilosis, Candida sojae, Candida sorboxylosa, Candida succiphila,Candida tenuis, Candida tropicalis, Candida utilis, Candida versatilis,Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Capnocytophagagingivalis, Capnocytophaga granulosa, Capnocytophaga haemolytica,Capnocytophaga ochracea, Capnocytophaga sputigena, Cardiobacteriumhominis, Cardiobacterium valvarum, Catabacter hongkongensis, Cedeceaneteri, Ceratocystis adiposa, Ceratocystis albifundus, Ceratocystiseucalypticola, Ceratocystis fimbriata, Ceratocystis manginecans,Ceratocystis platani, Cercospora fijiensis, Chaetomium globosum,Chaetomium thermophilum, Chlamydia psittaci Chlamydophila psittaci>,Chlamydia trachomatis, Chlamydophila pneumoniae, Chromobacteriumviolaceum, Chryseobacterium gleum, Chryseobacterium indologenes,Chrysosporium queenslandicum, Citrobacter amalonaticus, Citrobacterfreundii, Citrobacter koseri, Cladophialophora bantiana,Cladophialophora carrionii, Cladophialophora immunda, Cladophialophorapsammophila, Cladophialophora yegresii, Clonorchis sinensis, Clostridiumbaratii, Clostridium bifermentans, Clostridium clostridioforme,Clostridium difficile, Clostridium innocuum, Clostridium novyi,Clostridium perfringens, Clostridium sordellii, Clostridium tetani,Coccidioides immitis, Coccidioides posadasii, Cokeromyces recurvatus,Colletotrichum acutatum, Colletotrichum falcatum, Colletotrichumfioriniae, Colletotrichum gloeosporioides, Colletotrichum godetiae,Colletotrichum graminicola, Colletotrichum higginsianum, Colletotrichumincanum, Colletotrichum nymphaeae, Colletotrichum orbiculare,Colletotrichum salicis, Colletotrichum simmondsii, Colletotrichumsublineola, Colletotrichum tofieldiae, Comamonas testosteroni,Conidiobolus coronatus, Conidiobolus incongruus, Coniosporium apollinis,Corynebacterium accolens, Corynebacterium afermentans, Corynebacteriumamycolatum, Corynebacterium argentoratense, Corynebacterium aurimucosum,Corynebacterium diphtheriae, Corynebacterium falsenii, Corynebacteriumfreiburgense, Corynebacterium freneyi, Corynebacterium glucuronolyticum,Corynebacterium jeikeium, Corynebacterium kroppenstedtii,Corynebacterium kutscheri, Corynebacterium lipophiloflavum,Corynebacterium lymphophilum, Corynebacterium massiliense,Corynebacterium matruchotii, Corynebacterium minutissimum,Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum,Corynebacterium pseudotuberculosis, Corynebacterium renale,Corynebacterium riegelii, Corynebacterium simulans, Corynebacteriumstations, Corynebacterium striatum, Corynebacterium timonense,Corynebacterium tuscaniense, Corynebacterium ulcerans, Corynebacteriumurealyticum, Corynebacterium xerosis, Corynespora cassiicola, Cowpoxvirus, Coxiella burnetii, Cryptococcus bacillisporus, Cryptococcusbestiolae, Cryptococcus dejecticola, Cryptococcus deuterogattii,Cryptococcus fagi, Cryptococcus gattii, Cryptococcus neoformans,Cryptococcus pinus, Cryptococcus skinneri, Cryptococcus tetragattii,Cryptosporidium hominis, Cryptosporidium muris, Cryptosporidium parvum,Cunninghamella bertholletiae, Cupriavidus gilardii, Cupriavidusmetallidurans, Curvularia lunata, Cyclospora cayetanensis, Cyphellophoraeuropaea, Cytomegalovirus (CMV), Debaryomyces fabryi, Delftiaacidovorans, Dermabacter hominis, Dermacoccus nishinomiyaensis,Diaporthe ampelina, Diaporthe aspalathi, Diaporthe longicolla,Dirofilaria immitis, Dracunculus medinensis, Dysgonomonascapnocytophagoides, Dysgonomonas gadei, Dysgonomonas hofstadii,Dysgonomonas mossii, Echinococcus granulosus, Echinococcusmultilocularis, Echinostoma caproni, Edwardsiella hoshinae, Edwardsiellatarda, Eggerthella lenta, Ehrlichia canis, Ehrlichia chaffeensis,Ehrlichia muris, Eikenella corrodens, Elizabethkingia anophelis,Elizabethkingia meningoseptica, Elizabethkingia miricola, Emmonsiacrescens, Emmonsia parva, Empedobacter brevis, Encephalitozoon cuniculi,Encephalitozoon hellem, Encephalitozoon intestinalis, Encephalitozoonromaleae, Entamoeba histolytica, Enterobacter aerogenes, Enterobacteramnigenus, Enterobacter cloacae complex, Enterobacter sakazakii,Enterobius vermicularis, Enterococcus asini, Enterococcus avium,Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae,Enterococcus dispar, Enterococcus durans, Enterococcus faecalis,Enterococcus faecium, Enterococcus gallinarum, Enterococcus gilvus,Enterococcus haemoperoxidus, Enterococcus hirae, Enterococcus italicus,Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pallens,Enterococcus phoeniculicola, Enterococcus pseudoavium, Enterococcusraffinosus, Enterococcus saccharolyticus, Enterococcus sulfureus,Enterococcus thailandicus, Enterocytozoon bieneusi, Epstein-Barr virus(EBV), Erysipelothrix rhusiopathiae, Escherichia albertii, Escherichiablattae, Escherichia coli, Escherichia fergusonii, Escherichiahermannii, Escherichia vulneris, Eubacterium limosum, Eubacteriumnodatum, Exophiala alcalophila, Exophiala aquamarina, Exophialacalicioides, Exophiala dermatitidis, Exophiala mesophila, Exophialaoligosperma, Exophiala sideris, Exophiala spinifera, Exophialaxenobiotica, Facklamia hominis, Facklamia sourekii, Fasciola hepatica,Filifactor alocis, Filobasidium wieringae, Finegoldia magna, Fonsecaeaerecta, Fonsecaea monophora, Fonsecaea multimorphosa, Fonsecaea nubica,Fonsecaea pedrosoi, Francisella hispaniensis, Francisella noatunensis,Francisella philomiragia, Francisella tularensis, Fusarium avenaceum,Fusarium circinatum, Fusarium fujikuroi, Fusarium graminearum, Fusariumlangsethiae, Fusarium nygamai, Fusarium oxysporum, Fusarium poae,Fusarium pseudograminearum, Fusarium sambucinum, Fusarium temperatum,Fusarium verticillioides, Fusarium virguliforme, Fusobacteriummortiferum, Fusobacterium necrophorum, Fusobacterium nucleatum,Fusobacterium varium, Gammapapillomavirus 1, Gammapapillomavirus 2,Gammapapillomavirus 3, Gammapapillomavirus 4, Gammapapillomavirus 5,Gammapapillomavirus 6, Gammapapillomavirus 7, Gammapapillomavirus 8,Gammapapillomavirus 9, Gammapapillomavirus 10, Gammapapillomavirus 11,Gammapapillomavirus 13, Gammapapillomavirus 14, Gammapapillomavirus 15,Gammapapillomavirus 16, Gammapapillomavirus 17, Gammapapillomavirus 19,Gardnerella vaginalis, Gemella bergeri, Gemella haemolysans, Gemellamorbillorum, Gemella sanguinis, Geotrichum candidum, Giardia lamblia,Gordonia bronchialis, Gordonia rubripertincta, Gordonia terrae,Gordonibacter pamelaeae, Granulicatella adiacens, Granulicatellaelegans, Grimontia hollisae, Haemophilus aegyptius, Haemophilus ducreyi,Haemophilus haemolyticus, Haemophilus influenzae, Haemophilusparahaemolyticus, Haemophilus parainfluenzae, Hafnia alvei,Hanseniaspora uvarum, Hansenula fabianii, Helicobacter cinaedi,Helicobacter fennelliae, Helicobacter pylori, Herpes B virus, Herpessimplex virus type 1 (HSV-1), Herpes simplex virus type 2 (HSV-2),Histoplasma capsulatum, Human adenovirus A, Human adenovirus B, Humanadenovirus C, Human adenovirus D, Human adenovirus E, Human adenovirusF, Human bocavirus, Human herpesvirus 6A, Human herpesvirus 6B, Humanherpesvirus 7, Human papillomavirus, Human papillomavirus 132-likeviruses, Human papillomavirus type 136, Human papillomavirus type 140,Human papillomavirus type 154, Human papillomavirus type 167, Humanparvovirus, Human polyomavirus 6, Human polyomavirus 7, Hymenolepisnana, JC polyomavirus, Kaposi sarcoma-associated herpesvirus, KIpolyomavirus, Kingella denitrificans, Kingella kingae, Kingella oralis,Klebsiella oxytoca, Klebsiella pneumoniae, Kluyvera ascorbata, Kluyveracryocrescens, Kluyvera intermedia, Kluyveromyces lactis, Kocuriakristinae, Kytococcus sedentarius, Lachancea kluyveri, Lachancealanzarotensis, Lachancea thermotolerans, Lachancea waltii, Lactobacillusacidophilus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillusfermentum, Lactobacillus gasseri, Lactobacillus iners, Lactobacillusjensenii, Lactobacillus plantarum, Lactobacillus rhamnosus,Lactobacillus sakei, Lactobacillus ultunensis, Lactococcus garvieae,Leclercia adecarboxylata, Legionella anisa, Legionella bozemanae,Legionella cherrii, Legionella drancourtii, Legionella dumoffii,Legionella fairfieldensis, Legionella fallonii, Legionella geestiana,Legionella hackeliae, Legionella jamestowniensis, Legionellalansingensis, Legionella longbeachae, Legionella massiliensis,Legionella micdadei, Legionella moravica, Legionella norrlandica,Legionella oakridgensis, Legionella pneumophila, Legionellashakespearei, Legionella wadsworthii, Leifsonia aquatica, Leishmaniaaethiopica, Leishmania amazonensis, Leishmania braziliensis, Leishmaniadonovani, Leishmania major, Leishmania mexicana, Leishmania panamensis,Leishmania peruviana, Leishmania tropica, Leminorella grimontii,Leptosphaeria maculans, Leptospira alexanderi, Leptospira alstonii,Leptospira biflexa, Leptospira borgpetersenii, Leptospira broomii,Leptospira fainei, Leptospira inadai, Leptospira interrogans, Leptospirakirschneri, Leptospira kmetyi, Leptospira licerasiae, Leptospiramayottensis, Leptospira meyeri, Leptospira noguchii, Leptospirasantarosai, Leptospira terpstrae, Leptospira vanthielii, Leptospiraweilii, Leptospira wolbachii, Leptospira wolffii, Leptospira yanagawae,Leptotrichia buccalis, Leuconostoc citreum, Leuconostoc lactis,Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lichtheimiacorymbifera, Lichtheimia ramosa, Listeria grayi, Listeria innocua,Listeria ivanovii, Listeria monocytogenes, Listeria seeligeri, Listeriawelshimeri, Loa loa, Lodderomyces elongisporus, Macrophomina phaseolina,Madurella mycetomatis, Malassezia caprae, Malassezia cuniculi,Malassezia dermatis, Malassezia equina, Malassezia furfur, Malasseziaglobosa, Malassezia nana, Malassezia obtusa, Malassezia pachydermatis,Malassezia slooffiae, Malassezia sympodialis, Malassezia yamatoensis,Mannheimia haemolytica, Megasphaera micronuciformis, Memnoniellaechinata, Merkel cell polyomavirus, Metarhizium acridum, Metarhiziumalbum, Metarhizium anisopliae, Metarhizium brunneum, Metarhiziumguizhouense, Metarhizium majus, Metarhizium rileyi, Metarhiziumrobertsii, Methanobrevibacter smithii, Metschnikowia bicuspidata,Metschnikowia fructicola, Microbacterium foliorum, Microbacteriumoxydans, Microbacterium paraoxydans, Microbacterium testaceum,Micrococcus luteus, Micrococcus lylae, Microsporum canis, Microsporumgypseum, Mobiluncus curtisii, Mobiluncus mulieris, Moellerellawisconsensis, Mogibacterium timidum, Molluscum contagiosum virus,Monkeypox virus, Moraxella atlantae, Moraxella catarrhalis, Moraxellalacunata, Moraxella nonliquefaciens, Moraxella phenylpyruvica,Morganella morganii, Mortierella alpina, Mortierella elongata,Mortierella verticillata, Mucor ambiguus, Mucor circinelloides, Mucorindicus, Mucor irregularis, Mucor velutinosus, Mupapillomavirus 1,Mupapillomavirus 2, Myceliophthora thermophila, Mycobacterium abscessus,Mycobacterium arupense, Mycobacterium asiaticum, Mycobacterium aviumcomplex (MAC), Mycobacterium brisbanense, Mycobacterium canariasense,Mycobacterium chelonae, Mycobacterium chimaera, Mycobacteriumcosmeticum, Mycobacterium fortuitum, Mycobacterium genavense,Mycobacterium goodii, Mycobacterium gordonae, Mycobacterium haemophilum,Mycobacterium heckeshornense, Mycobacterium heraklionense, Mycobacteriumimmunogenum, Mycobacterium iranicum, Mycobacterium kansasii,Mycobacterium kumamotonense, Mycobacterium kyorinense, Mycobacteriumleprae, Mycobacterium mageritense, Mycobacterium malmoense,Mycobacterium marinum, Mycobacterium nebraskense, Mycobacteriumneoaurum, Mycobacterium novocastrense, Mycobacterium parascrofulaceum,Mycobacterium peregrinum, Mycobacterium phlei, Mycobacteriumscrofulaceum, Mycobacterium senegalense, Mycobacterium septicum,Mycobacterium setense, Mycobacterium simiae, Mycobacterium smegmatis,Mycobacterium szulgai, Mycobacterium thermoresistibile, Mycobacteriumtriplex, Mycobacterium tuberculosis complex, Mycobacterium tusciae,Mycobacterium vaccae, Mycobacterium wolinskyi, Mycobacterium xenopi,Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis,Mycoplasma hyopneumoniae, Mycoplasma penetrans, Mycoplasma pneumoniae,Mycoplasma pulmonis, Myroides odoratimimus, Myroides odoratus, Naegleriafowleri, Nakaseomyces bacillisporus, Nakaseomyces delphensis, Nakazawaeapeltata, Necator americanus, Nectria haematococca, Neisseria elongata,Neisseria flavescens, Neisseria gonorrhoeae, Neisseria lactamica,Neisseria meningitidis, Neisseria mucosa, Neisseria polysaccharea,Neisseria sicca, Neisseria weaveri, Neofusicoccum parvum, Neorickettsiahelminthoeca, Neorickettsia sennetsu, Nocardia abscessus, Nocardiaacidovorans, Nocardia africana, Nocardia alba, Nocardia amamiensis,Nocardia anaemiae, Nocardia aobensis, Nocardia araoensis, Nocardiaarthritidis, Nocardia asiatica, Nocardia beijingensis, Nocardiabrasiliensis, Nocardia brevicatena, Nocardia caishijiensis, Nocardiacarnea, Nocardia cerradoensis, Nocardia concava, Nocardia coubleae,Nocardia crassostreae, Nocardia cummidelens, Nocardia cyriacigeorgica,Nocardia dassonvillei, Nocardia elegans, Nocardia exalbida, Nocardiafarcinica, Nocardia flavorosea, Nocardia fusca, Nocardia gamkensis,Nocardia grenadensis, Nocardia harenae, Nocardia higoensis, Nocardiaignorata, Nocardia inohanensis, Nocardia jejuensis, Nocardiajiangxiensis, Nocardia kruczakiae, Nocardia lijiangensis, Nocardiamexicana, Nocardia mikamii, Nocardia miyunensis, Nocardia niigatensis,Nocardia niwae, Nocardia nova, Nocardia otitidiscaviarum, Nocardiapaucivorans, Nocardia pneumoniae, Nocardia pseudobrasiliensis, Nocardiapseudovaccinii, Nocardia puris, Nocardia rhamnosiphila, Nocardiasalmonicida, Nocardia seriolae, Nocardia shimofusensis, Nocardiasienata, Nocardia soli, Nocardia speluncae, Nocardia takedensis,Nocardia tenerifensis, Nocardia terpenica, Nocardia testacea, Nocardiathailandica, Nocardia transvalensis, Nocardia uniformis, Nocardiavaccinii, Nocardia vermiculata, Nocardia veterana, Nocardia vinacea,Nocardia violaceofusca, Nocardia xishanensis, Nocardia yamanashiensis,Nosema apis, Nosema bombycis, Nosema ceranae, Nupapillomavirus 1,Ochrobactrum anthropi, Ochrobactrum intermedium, Ochroconis constricta,Ochroconis gallopava, Odoribacter splanchnicus, Oerskovia turbata,Ogataea methanolica, Ogataea parapolymorpha, Ogataea polymorpha,Oligella ureolytica, Oligella urethralis, Olsenella uli, Onchocercavolvulus, Ophiostoma novo-ulmi, Ophiostoma piceae, Opisthorchisviverrini, Orf virus, Oribacterium sinus, Orientia tsutsugamushi,Paecilomyces hepiali, Paenibacillus alvei, Pantoea agglomerans,Paraburkholderia fungorum, Paracoccidioides brasiliensis,Paracoccidioides lutzii, Parvimonas micra, Pasteurella bettyae,Pasteurella multocida, Pasteurella pneumotropica, Pediococcusacidilactici, Pediococcus pentosaceus, Penicillium brasilianum,Penicillium camemberti, Penicillium capsulatum, Penicillium carneum,Penicillium digitatum, Penicillium expansum, Penicillium freii,Penicillium griseofulvum, Penicillium islandicum, Penicillium italicum,Penicillium marneffei, Penicillium nalgiovense, Penicillium nordicum,Penicillium oxalicum, Penicillium paneum, Penicillium paxilli,Penicillium piceum, Penicillium pinophilum, Penicillium purpurogenum,Penicillium roqueforti, Penicillium rubens, Penicillium verruculosum,Peptoniphilus coxii, Peptoniphilus duerdenii, Peptoniphilus harei,Peptoniphilus indolicus, Peptoniphilus lacrimalis, Peptoniphilusrhinitidis, Peptostreptococcus anaerobius, Peptostreptococcus stomatis,Phaeoacremonium minimum, Phanerochaete carnosa, Phanerochaetechrysosporium, Phellinus noxius, Phialophora attae, Phoma herbarum,Photobacterium damselae, Photorhabdus asymbiotica, Photorhabdusluminescens, Phycomyces blakesleeanus, Pichia anomala, Plasmodiumcynomolgi, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium ovale,Plasmodium vivax, Plesiomonas shigelloides, Pluralibacter gergoviae,Pneumocystis carinii, Pneumocystis jirovecii, Pneumocystis murina,Porcine circovirus 1, Porcine circovirus 2, Porphyromonasasaccharolytica, Porphyromonas gingivalis, Prevotella bivia, Prevotellabuccae, Prevotella buccalis, Prevotella corporis, Prevotella denticola,Prevotella disiens, Prevotella intermedia, Prevotella loescheii,Prevotella melaninogenica, Prevotella oralis, Primate bocaparvovirus 1,Propionibacterium acidifaciens, Propionibacterium granulosum,Propionibacterium propionicum, Proteus mirabilis, Proteus vulgaris,Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii,Pseudocowpox virus, Pseudomonas aeruginosa, Pseudomonas alcaligenes,Pseudomonas fluorescens, Pseudomonas fulva, Pseudomonas luteola,Pseudomonas mendocina, Pseudomonas mosselii, Pseudomonas oryzihabitans,Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudoramibacteralactolyticus, Pseudozyma hubeiensis, Purpureocillium lilacinum,Pyrenochaeta lycopersici, Pyrenochaeta mackinnonii, Rahnella aquatilis,Ralstonia insidiosa, Ralstonia mannitolilytica, Ralstonia pickettii,Ramichloridium mackenziei, Rasamsonia emersonii, Rhizoctonia solani,Rhizomucor miehei, Rhizomucor variabilis, Rhizopus delemar, Rhizopusmicrosporus, Rhizopus oryzae, Rhizopus stolonifer, Rhodococcus equi,Rhodococcus erythropolis, Rhodococcus fascians, Rhodococcus rhodochrous,Rhodotorula graminis, Rhodotorula mucilaginosa, Rhodotorula toruloides,Rhytidhysteron rufulum, Rickettsia akari, Rickettsia amblyommii,Rickettsia australis, Rickettsia canadensis, Rickettsia conorii,Rickettsia felis, Rickettsia helvetica, Rickettsia honei, Rickettsiajaponica, Rickettsia massiliae, Rickettsia monacensis, Rickettsiaparkeri, Rickettsia prowazekii, Rickettsia raoultii, Rickettsiarickettsii, Rickettsia sibirica, Rickettsia slovaca, Rickettsia typhi,Riemerella anatipestifer, Roseomonas cervicalis, Roseomonas fauriae,Roseomonas gilardii, Roseomonas mucosa, Rothia aeria, Rothiadentocariosa, Rothia mucilaginosa, Saccharomyces cerevisiae, Saksenaeaoblongispora, Saksenaea vasiformis, Salmonella bongori, Salmonellaenterica, Scedosporium apiospermum, Scedosporium aurantiacum,Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni,Schizophyllum commune, Serratia ficaria, Serratia fonticola, Serratialiquefaciens, Serratia marcescens, Serratia plymuthica, Serratiarubidaea, Shewanella algae, Shewanella putrefaciens, Shigella boydii,Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Slackiaexigua, Solobacterium moorei, Sphingobacterium spiritivorum,Sporopachydermia quercuum, Sporothrix brasiliensis, Sporothrix globosa,Sporothrix insectorum, Sporothrix pallida, Sporothrix schenckii,Stachybotrys chartarum, Stachybotrys chlorohalonata, Staphylococcusagnetis, Staphylococcus arlettae, Staphylococcus aureus, Staphylococcusauricularis, Staphylococcus capitis, Staphylococcus caprae,Staphylococcus carnosus, Staphylococcus caseolyticus, Staphylococcuschromogenes, Staphylococcus cohnii, Staphylococcus condimenti,Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcusgallinarum, Staphylococcus haemolyticus, Staphylococcus hominis,Staphylococcus hyicus, Staphylococcus lentus, Staphylococcuslugdunensis, Staphylococcus pasteuri, Staphylococcus pettenkoferi,Staphylococcus pseudintermedius, Staphylococcus saprophyticus,Staphylococcus schleiferi, Staphylococcus sciuri, Staphylococcus simiae,Staphylococcus simulans, Staphylococcus succinus, Staphylococcusvitulinus, Staphylococcus warneri, Staphylococcus xylosus, Stemphyliumlycopersici, STL polyomavirus, Streptobacillus moniliformis,Streptococcus agalactiae, Streptococcus anginosus, Streptococcus canis,Streptococcus constellatus, Streptococcus cricetus, Streptococcuscristatus, Streptococcus dysgalactiae, Streptococcus equi, Streptococcusequinus, Streptococcus ferus, Streptococcus gallolyticus, Streptococcusgordonii, Streptococcus hyovaginalis, Streptococcus infantarius,Streptococcus infantis, Streptococcus iniae, Streptococcus intermedius,Streptococcus lutetiensis, Streptococcus macacae, Streptococcusmassiliensis, Streptococcus mitis, Streptococcus mutans, Streptococcusoralis, Streptococcus parasanguinis, Streptococcus pasteurianus,Streptococcus peroris, Streptococcus pneumoniae, Streptococcus porcinus,Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcusratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcussobrinus, Streptococcus suis, Streptococcus thermophilus, Streptococcusuberis, Streptococcus vestibularis, Streptomyces somaliensis,Strongyloides stercoralis, Sutterella wadsworthensis, Syncephalastrummonosporum, Syncephalastrum racemosum, Taenia asiatica, Talaromycescellulolyticus, Talaromyces leycettanus, Talaromyces stipitatus, Tanapoxvirus, Tatumella ptyseos, Thermoascus crustaceus, Thermomyceslanuginosus, Thielavia terrestris, Torque teno virus, Torque teno virus1, Torque teno virus 2, Torque teno virus 3, Torque teno virus 4, Torqueteno virus 6, Torque teno virus 7, Torque teno virus 8, Torque tenovirus 10, Torque teno virus 12, Torque teno virus 14, Torque teno virus15, Torque teno virus 16, Torque teno virus 19, Torque teno virus 25,Torque teno virus 26, Torque teno virus 27, Torque teno virus 28,Torulaspora delbrueckii, Toxocara canis, Toxoplasma gondii,Trachipleistophora hominis, Treponema pallidum, Trichinella nelsoni,Trichinella pseudospiralis, Trichinella spiralis, Trichodermaasperellum, Trichoderma atroviride, Trichoderma gamsii, Trichodermahamatum, Trichoderma harzianum, Trichoderma longibrachiatum, Trichodermaparareesei, Trichoderma reesei, Trichoderma virens, Trichodysplasiaspinulosa-associated polyomavirus, Trichomonas vaginalis, Trichophytonbenhamiae, Trichophyton interdigitale, Trichophyton rubrum, Trichophytonverrucosum, Trichosporon asahii, Trichosporon cutaneum, Trichosporonguehoae, Trichosporon oleaginosus, Trichosporon porosum, Trichuristrichiura, Tropheryma whipplei, Trypanosoma brucei, Trypanosoma cruzi,Tsukamurella paurometabola, Turicella otitidis, Ureaplasma parvum,Ureaplasma urealyticum, Ustilago cynodontis, Ustilago esculenta,Ustilago hordei, Ustilago maydis, Ustilago trichophora, Vaccinia virus,Valsa mali, Varicella-zoster virus (VZV), Variola virus, Veillonelladispar, Veillonella montpellierensis, Veillonella parvula, Verticilliumalfalfae, Verticillium dahliae, Verticillium longisporum, Verticilliumtricorpus, Vibrio alginolyticus, Vibrio cholerae, Vibrio fluvialis,Vibrio furnissii, Vibrio harveyi, Vibrio metschnikovii, Vibrio mimicus,Vibrio parahaemolyticus, Vibrio vulnificus, Vittaforma corneae,Volvariella volvacea, Wallemia ichthyophaga, Wallemia mellicola,Weeksella virosa, Weissella confusa, Weissella paramesenteroides,Wickerhamomyces ciferrii, Wolbachia pipientis, WU Polyomavirus,Wuchereria bancrofti, Xanthomonas axonopodis, Yaba monkey tumor virus,Yarrowia deformans, Yarrowia keelungensis, Yarrowia lipolytica, Yersiniaenterocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersiniakristensenii, Yersinia pestis, Yersinia pseudotuberculosis, Yersiniaruckeri, Yokenella regensburgei. In some emboiments, the pathogens arecell-free. In some emboiments, the pathogen are intact, in exosomes, orassociated with an exosome.

XII. Kits

The methods and composition of the disclosure can also be supplied inform of a kit. In general, a kit comprises a set of instructions forcarrying out one or more methods of present disclosure.

In general, a kit provides concurrent detection of different nucleicacid forms in a sample. For example, in some embodiments, a kit canprovide for the concurrent analysis of 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore nucleic acid forms in a sample. In another embodiment, a kitprovides for the detection and processing of only one nucleic acid formin a sample.

In some embodiments, the kit can be tailored for a specific applicationsuch as diagnosis, prognosis, prediction of a disease, drug response,infection, fetal health information, or analysis of various geneticmutations related to a specific condition or disease. In someembodiments, the kit can be tailored with additional reagents orconsumables for use with specific sample types, such as, blood, bodyfluids, tissues, particular cell types, or isolated nucleic acids.

The kit can be tailored for different detection methods (e.g.,microarray, qPCR, ddPCR, or sequencing as provided herein). Depending ondetection method used the kit can comprise the particular hardware,software, or reagents required for detection.

The kit can also comprise instructions. In some embodiments, theinstructions of the kit outline steps for the detection and processingof highly degraded DNA or RNA or cell-free samples. In some embodiments,the instructions of the kit outline steps for the detection andprocessing of sample having or at risk of having disease or infection.

EXAMPLES Example 1: Concurrent Analysis of Nucleic Acids Using thePrimer Extension Method

The study was conducted to test the primer extension method for theconcurrent detection of different nucleic acids forms in a sample usingpolymerases that have different preferences for DNA and RNA templates.

A 10 μL sample was obtained that contained a mixture of RNA and DNA.

Uracil Excision and DNA Cleavage at Abasic Sites.

In an initial optional step, abasic sites were removed usingEndonuclease VIII. In addition, deoxyuracils may optionally be removedfrom the nucleic acids in order to improve sequence accuracy. For eachsample, a reaction mixture was prepared with a total volume of 42 μL in0.5 mL tubes. The reaction mixture includes water (to 42 μL), 10×CircLigase buffer II (8 μL), MnCl₂ (4 μL, 50 mM), DNA extract (max. 29μL), Endonuclease VIII (0.5 μL, 10 U μL⁻¹), and optionally Afu UDG (0.5μL, 2 U μL⁻¹). The tubes were mixed and spun in a microcentrifuge. Thereactions were incubated in a thermal cycler with a heated lid for 1 hrat 37° C.

Dephosphorylation and Heat Denaturation.

Before denaturation, phosphatase was added to the sample in order toremove residual phosphate groups from the 5′ and 3′ ends of the DNAstrands in order to minimize self-circularization and prevent thephosphate groups from interfering with adapter ligation. FastAP (1 μL, 1U) was added to each reaction mixture. Tubes were mixed and spun brieflyin a microcentrifuge. The total reaction volume was 43 μL. The reactionswere incubated in a thermal cycler with a heated lid for 10 min at 37°C., and then at 95° C. for 2 min. While the thermal cycler was still at95° C., the tubes were quickly transferred into an ice-water bath. Thereaction mix was cooled down for at least 1 min. The tubes were spunbriefly in a microcentrifuge and placed in a tube rack at roomtemperature.

Ligation of the First Adapter.

PEG-4000 (32 μL, 50%), single-stranded adapter oligo CL78 (1 μL, 10 μM,5′-[Phosphate]AGATCGGAAG[C3Spacer]₁₀[TEG-biotin]-3′, (TEG=triethyleneglycol spacer)), and CircLigase II (4 μL, 100 U μL⁻¹) were added to thereaction mixtures to obtain a final reaction volume of 80 μL. Thecontents of the tubes were mixed before adding CircLigase II. The tubeswere spun briefly in a microcentrifuge. The reaction mixtures wereincubated in a thermal cycler with a heated lid for 1 hr at 60° C. Stopsolution (2 μL) (98 μL of 0.5 M EDTA (pH 8.0) and 2 μL of Tween 20 werecombined to make 100 μL of stop solution) was added to each reactionmixture. The contents were mixed, and the tubes were spun in amicrocentrifuge.

Immobilization of Ligation Products on Beads.

The ligation products containing the biotinylated adapters may beimmobilized on streptavidin beads. Such immobilization may be useful forwash steps. The stock of Dynabeads MyOne streptavidin C1 beads (LifeTechnologies) were resuspended by vortexing. For each sample, the beadsuspension (20 μL) was transferred into a 1.5-mL tube. The beads werepelleted using a magnetic rack. The supernatant was discarded, and thebeads were washed twice with bead-binding buffer (500 μL). 7.63 mL ofwater (HPLC-grade), 2 mL of 5 M NaCl, 100 μL of 1 M Tris-HCl (pH 8.0),20 μL of 0.5 M EDTA (pH 8.0), 5 μL of Tween 20, and 250 μL of 20%(wt/vol) SDS were combined to make 10 mL of bead-binding buffer. SDS wasadded immediately before use. The beads were resuspended in a volume ofbead-binding buffer corresponding to the number of samples times 250 μL(e.g., 1 mL for four samples). Per sample, an aliquot of 250 μL of beadsuspension was transferred to a 1.5-mL tube. The ligation reactions wereincubated for 1 min at 95° C. in a thermal cycler with a heated lid.While the thermal cycler was still at 95° C., the tubes were quicklytransferred into an ice-water bath. The reaction mixture was cooled downfor at least 1 min. The tubes were spun briefly in a microcentrifuge.The ligation reactions were added to the bead suspensions. The tubeswere rotated for 20 min at room temperature. The tubes were spun brieflyin a microcentrifuge. The beads were pelleted using a magnetic rack, andthe supernatant was discarded. The beads were washed once with 200 μL ofwash buffer A and once with 200 μL of wash buffer B. 47.125 mL of water,1 mL of 5 M NaCl, 500 μL of 1 M Tris-HCl (pH 8.0), 100 μL of 0.5 M EDTA(pH 8.0), 25 μL of Tween 20, and 1.25 mL of 20% (wt/vol) SDS werecombined to make 50 mL of wash buffer A, 48.375 mL of water, 1 mL of 5 MNaCl, 500 μL of 1 M Tris-HCl (pH 8.0), 100 μL of 0.5 M EDTA (pH 8.0),and 25 μL of Tween 20 were combined to make 50 mL of wash buffer B.

Primer Annealing and Extension.

For this step, a primer complementary to the adapter was used to copythe template strand. A master mix was prepared for the required numberof reactions, according to the manufacturer's instructions (47 μL perreaction).

Bst 2.0 Polymerase.

For the master mix comprising, Bst 2.0 polymerase, 40.5 μL water, 5 μL10× isothermal amplification buffer (New England Biolabs), 0.5 μL dNTPmix (25 mM each), and 1 μL extension primer CL9(5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′, 100 μM) were combined to make47 μL master mix. The beads were pelleted using a magnetic rack, and thewash buffer was discarded. The 47-μL reaction mixture was added to thepelleted beads, and the beads were resuspended by vortexing. The tubeswere incubated in a thermal shaker for 2 min at 65° C. The tubes wereplaced in an ice-water bath for 1 min and then were immediatelytransferred to a thermal cycler precooled to 15° C. While the tubes wereplaced on the thermal cycler, a polymerase (e.g., Bst 2.0 polymerase (3μL, 24 U, New England Biolabs), DNA polymerase, or reversetranscriptase) was added to each reaction mixture. The tubes were mixedbriefly by vortexing and returned to the thermal cycler. The reactionmixtures were incubated by increasing the temperature by 1° C. perminute, ramping the temperature up from 15° C. to 37° C. The reactionmixtures were incubated for 5 min at 37° C. The tubes were spun brieflyin a microcentrifuge. The beads were pelleted using a magnetic rack, andthe supernatant was discarded.

The beads were washed once with 200 μl of wash buffer A. The beads wereresuspended in 100 μL of stringency wash buffer (49.5 ml of water, 250μl of 20% (wt/vol) SDS, and 250 μL of 20×SSC buffer were combined tomake 50 mL of stringency wash buffer), and the bead suspensions wereincubated for 3 min at 45° C. in a thermal shaker. The beads werepelleted using a magnetic rack, and the supernatant was discarded. Thebeads were washed once with 200 μL of wash buffer B.

SMARTer RT.

Clonetech SMARTer RT can be used in place of Bst 2.0 polymerase,according to the manufacturer's instructions with the followingmodifications: 1) in-house extension primer CL9(5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′) was used for the primerextension in step 4 of the protocol in place of the manufacturer's 3′SMART CDS Primer II A, and 2) SMART-Seq v4 oligonucleotide was not usedin the Step 6 of the manufacturer's protocol. The beads were pelleted onthe magnet and reverse transcription products were collected in thesupernatant, purified, amplified by PCR, and sequenced. Results werequantified also using TapeStation (Agilent).

If the sample was processed with Bst 2.0 polymerse in the primerextension step then the beads were washed once with 200 μl of washbuffer A. The beads were resuspended in 100 μL of stringency wash buffer(49.5 ml of water, 250 μl of 20% (wt/vol) SDS, and 250 μL of 20×SSCbuffer were combined to make 50 mL of stringency wash buffer), and thebead suspensions were incubated for 3 min at 45° C. in a thermal shaker.The beads were pelleted using a magnetic rack, and the supernatant wasdiscarded. The beads were washed once with 200 μL of wash buffer B.

Blunt-End Repair.

A blunt-end repair step may be used, particularly when blunt-enddouble-stranded second adapters were appended to the nucleic acid, whichgenerally occurs at the end opposite to the end of the nucleic acid towhich the first adapter was appended. A master mix was prepared for therequired number of reactions (99 μL per reaction). 86.1 μL water, 10 μL10× Buffer Tango (Thermo Scientific), 2.5 μL Tween 20 (1%), and 0.4 μLdNTP (25 mM each) were combined to make 99 μL master mix. The beads werepelleted using a magnetic rack, and the wash buffer was discarded. Thereaction mixture (99 μL) was added to the pelleted beads, and the beadswere resuspended by vortexing. T4 DNA polymerase (1 μL, 5 U, ThermoScientific) was added. The tubes were mixed briefly by vortexing. Thereaction mixtures were incubated for 15 min at 25° C. in a thermalshaker. The beads were kept suspended during incubation. EDTA (10 μL,0.5 M) was added to each reaction mixture and mixed by vortexing. Thebeads were pelleted using a magnetic rack, and the supernatant wasdiscarded. The beads were washed once with 200 μl of wash buffer A. Thebeads were resuspended in 100 μL of stringency wash buffer, and the beadsuspensions were incubated for 3 min at 45° C. in a thermal shaker. Thebeads were pelleted using a magnetic rack, and the supernatant wasdiscarded. The beads were washed once with 200 μL of wash buffer B.

Ligation of Second Adapter and Library Elution.

A master mix was prepared for the required number of reactions (98 μLper reaction). 73.5 μL water, 10 μL 10× T4 DNA ligase buffer, 10 μLPEG-4000 (50%), 2.5 μL Tween 20 (1%), and 2 μL double-stranded adapter(100 μM) were combined to make 98 μL master mix. To make thedouble-stranded adapter stock solution, 9.5 μL of TE buffer, 0.5 μL of 5M NaCl, 20 μL of 500 μM oligonucleotide CL53 (5′-CGACGCTCTTC-ddC)(ddC=dideoxycytidine), and 20 μL of 500 μM oligonucleotide CL73(5′-[Phosphate]GGAAGAGCGTCGTGTAGGGAAAGAG*T*G*T*A-3′) (*=phosphothioatelinkage) were combined in a PCR tube, the reaction mixture was incubatedin a thermal cycler for 10 s at 95° C., the temperature was slowlydecreased at the rate of 0.1° C. per second until reaching 14° C., and50 μL of TE buffer was added to the hybridized adapter to obtain aconcentration of 100 μM in a total volume of 100 μL. 49.4 mL of water,500 μL of 1 M Tris-HCl (pH 8.0), and 100 μL of 0.5 M EDTA (pH 8.0) werecombined to make 50 mL of TE buffer. The beads were pelleted using amagnetic rack, and the wash buffer was discarded. The reaction mixture(98 μL) was added to the pelleted beads, and the beads were resuspendedby vortexing. T4 DNA ligase (2 μL, 10 U, Thermo Scientific) was added.The contents were mixed briefly by vortexing. The reaction mixtures wereincubated for 1 hr at room temperature. The beads were kept suspendedduring incubation. The beads were pelleted using a magnetic rack, andthe supernatant was discarded. The beads were washed once with 200 μl ofwash buffer A. The beads were resuspended in 100 μL of stringency washbuffer, and the bead suspensions were incubated for 3 min at 45° C. in athermal shaker. The beads were pelleted using a magnetic rack, and thesupernatant was discarded. The beads were washed once with 200 μL ofwash buffer B. The beads were pelleted using a magnetic rack, and thesupernatant was discarded. TET buffer (25 μL) (49.375 mL of water, 500μL of 1 M Tris-HCl, 100 μL of 0.5 M EDTA, and 25 μL of Tween 20 werecombined to make 50 mL of TET buffer) was added to the pelleted beads,and the beads were resuspended by vortexing. The bead suspension wastransfer to 0.2-mL PCR strip tubes. The tubes were spun briefly in amicrocentrifuge. The bead suspensions were incubated for 1 min at 95° C.in a thermal cycler with a heated lid. The PCR strip tubes wereimmediately transferred to a 96-well magnetic rack. The supernatant wastransferred, which contains the library molecules, to a fresh 0.5-mLtube.

Library Amplification and Indexing.

A PCR mix was prepared using a unique combination of indexing primersfor each sample. The PCR mix can be prepared with 57 μL water (to 100μL), 10 μL 10× AccuPrime Pfx reaction mix (Life Technologies), 4 μL P7indexing primer (10 μM), 4 μL P5 indexing primer or IS4 (10 μM), 24 μLlibrary, and 1 μL AccuPrime Pfx polymerase (2.5 U μl⁻¹) (LifeTechnologies). The reactions were incubated in a thermal cycler with aninitial denaturation at 95° C. for 2 min, followed by a selected numberof PCR cycles, involving denaturation for 15 s at 95° C., annealing for30 s at 60° C. and primer extension for 1 min at 68° C. The amplifiedlibraries were purified using the MinElute PCR purification kit (Qiagen)or AMPure XP SPRI beads (Beckman Coulter) according to themanufacturer's instructions. The DNA was eluted in 20 μL of TE buffer.The fragment size distributions and concentrations of the DNA librarieswere determined by running the Agilent Bioanalyzer 2100 with a DNA 1000chip.

Sequencing.

For sequencing, the protocols and instructions for multiplex sequencingprovided by Illumina were followed. The sequencing primer of the firstread was replaced by the custom primer CL72(5′-ACACTCTTTCCCTACACGACGCTCTTCC-3′). A ready-to-use dilution of CL72was freshly prepared before sequencing by mixing 10 μL from the 100 μMstock solution with 1,990 μL of hybridization buffer (provided with thesequencing reagents).

FIG. 11 shows bar graphs comparing the DNA and RNA input of the startingsample with the final DNA and RNA output detected after sequencing. Bothpolymerases showed the ability to carry out the primer extension againstboth DNA and RNA substrates, FIG. 11. The Bst 2.0 DNA polymerase showedprimer extension for both templates, but preferentially worked on a DNAsubstrate, even in the presence of relatively high quantities of RNAsubstrate (FIG. 11B). However, the SMARTer reverse transcriptaseprovided primer extension against both DNA and RNA templates (FIG. 11A).

Example 2: Efficiency of Ligases in a Single Reaction Mixture

The study was conducted to determine if ligases can perform in a singlereaction mixture provided by the disclosure.

Different ligases were tested for their ligation efficiency toward RNAfragments in the presence of DNA. Three different ligases were tested,CircLigase II, Thermostable AppDNA/RNA ligase, and T4 RNA ligase 1, intwo sample mixtures that each included 4.5 pM of a 100 bp DNAoligonucleotide and either 10 nM (high) or 0.1 nM (low) of a 50nucleotide RNA oligonucleotide. Following the ligation, the reactionmixtures were subjected to the protocol shown in FIG. 1 starting at theprimer extension reaction 130.

Preparation of RNA/DNA Master Mixes:

Master mixes with either “high” or “low” concentration of a RNAoligonucleotides were made. The “high” concentration master mixcontained RNA oligonucleotides at 10 nM, and the “low” concentrationmaster mix contained RNA oligonucleotides at 100 pM. Both master mixescontained DNA oligonucleotides at 4.5 pM.

Enzymes were used according to the manufacturer's instructions. Prior toligation, RNA oligonucleotides were treated with FastAP enzyme. DNAoligonucleotides were added after the FastAP treatment with therespective ligase.

The Adapter Used in the CircLigase II and T4 RNA Ligase 1 Ligation:

The adapter used was/5Phos/AGATCGGAAG/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/iSpC3/3BioTEG/ and contains 5′-phosphylation, 3′-biotinylation, and non-nucleotidepolymer extension. The Thermostable App ligase requires an Appmodification (e.g., pre-adenylation) at the 5′-end.

Ligation of the Adapter by CircLigase II:

RNA/DNA master mix (10 μL), deionized water (20 μL), CircLigase IIBuffer (8 μL), MnCl₂ (4 μL, 50 mM), and FastAP (1 μL) were mixed tophosphorylate the 5′-end and dephosphorylate the 3′-ends in RNA and DNAoligonucleotides with the FastAP kinase. Each reaction mix was incubatedat 37° C. for 10 min, followed by thermal deactivation at 95° C. for 2min, and immediately placed on ice before proceeding to the ligationstep. 50 w/v % PEG4000 (32 adapter oligo (1 and CircLigase II (4 μL)were added to the reaction from the kinase step to obtain a reactionmixture with 1× CircLigase Buffer II and 2.5 mM MnCl₂. The mixture wasincubated at 60° C. for 1 h, followed by the addition of Stop Solution(0.5 M EDTA pH 8.0, 2 v/v % Tween-20) (2 μL) and inactivation at 95° C.for 1 min. After, the mixture was placed on ice and purified using aZymo RNA purification column.

Ligation of the Adapter by Themostable AppDNA/RNA Ligase (NEB):

RNA/DNA master mix (10 deionized water (2.5 NEB Buffer #1 (2.0 MnCl₂ (2μL, 50 mM), and FastAP (1 μL) were mixed to phosphorylate the 5′-end anddephosphorylate the 3′-ends in RNA and DNA oligonucleotides with theFastAP kinase. Each reaction mix was incubated at 37° C. for 10 min,followed by thermal deactivation at 95° C. for 2 min, and immediatelyput on ice before proceeding to the ligation step. ThermostableAppDNA/RNA ligase (2.0 μL) and adapter oligo (0.5 μL) were added to thereaction from the kinase step to obtain a reaction mixture with 1× NEBBuffer #1 and 5 mM MnCl₂. The mixture was incubated at 65° C. for 1 hand inactivated at 90° C. for 3 min. The mixture was placed on ice andpurified with a Zymo RNA purification column.

Ligation of the Adapter by T4 RNA Ligase 1 (NEB):

RNA/DNA master mix (10 μL), deionized water (27 μL), 10× T4 RNA LigaseBuffer (6 μL), and FastAP (1 μL) were mixed to phosphorylate the 5′-endand dephosphorylate the 3′-ends in RNA and DNA oligonucleotides with theFastAP kinase. Each reaction mix was incubated at 37° C. for 10 min,followed by thermal deactivation at 95° C. for 2 min, and immediatelyput on ice before proceeding to the ligation step. ATP (6 μL, 10 mM), T4RNA ligase 1 (1 μL), and adapter oligo (3 μL) were added to the reactionfrom the kinase step to obtain a reaction mixture with 1× T4 RNA LigaseReaction Buffer and 1 mM ATP. The mixture was incubated at 37° C. for 1h, placed on ice, and purified with a Zymo RNA purification column.

Post-Ligation Reverse Transcription was Performed with Clonetech SMARTerRT:

The reverse transcription reaction was carried out according to themanufacturer's instructions with the following modifications: 1)in-house extension primer CL9 (5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′)was used for the primer extension reaction in step 4 of the protocol inplace of the manufacturer's 3′ SMART CDS Primer II A, and 2) SMART-Seqv4 oligonucleotide was not used in the step 6 of the manufacturer'sprotocol. Reverse transcription products were amplified by PCR. Theamplified products were quantified using TapeStation (Agilent).

FIG. 10 shows the detected and amplified products using gelelectrophoresis. The three tested ligases showed varying degrees ofefficacy in the presence of DNA. The arrow in FIG. 10 indicates theexpected 84 nt product (50 nt sequence and 34 nt adapter). TheCircLigase II showed the greatest efficiency for both high and low RNAconcentration mixtures (lanes B2 (high) and C2 (low)), with decreasingefficiency ligation for the thermostable App-DNA/RNA ligase (lanes D2(high) and E2 (low)), and for T4 RNA ligase 1 (lanes F2 (high) and G2(low)).

The CircLigase in lane B2 yielded 243 nM solution of reverse-transcribedRNA oligonucleotide after 11 cycles of PCR following the SMARTer RTstep, which is about 14.2% of the expected recovery.

Example 3: Efficiency of DNA and RNA Polymerases in a Single ReactionMixture

The study was conducted to determine if polymerase enzymes can performin the single reaction mixtures provided by the disclosure.

The ligation method was carried out on both DNA and RNA samples to theend of the primer extension step of FIG. 1, 130. Two differentpolymerases, Bst-polymerase and SMARTer reverse transcriptase weretested on the ligation products. Both polymerases were added at similarlevels to different single reaction mixtures comprising eight DNAfragments of different lengths, a single sample index spike-in DNAfragment, and 10 nM of RNA oligonucleotides.

FIG. 12 shows the detected and amplified products of the ligation methodusing gel electrophoresis. In FIG. 12, “L” indicates the molecularladder (25 bp Step Ladder). Lane, “1” shows the DNA detected in thesample using ligation and a Bst 2.0 polymerase. Lane “2” shows the RNAdetected in the sample using ligation and a SMARTer ReverseTranscriptase.

Example 4: Efficiency of Reverse Transcriptases in a Single ReactionMixture

The study was conducted to determine if reverse transcriptase enzymescan perform in the single reaction mixtures provided by the disclosure.

Different reverse transcriptase enzymes were tested for theirdiscrimination between DNA and RNA templates during the firstreplication reaction. Four different reverse transcriptases, SuperscriptIV RT (Invitrogen), M-MLV Rnase H(−) (Promega), SMARTer reversetranscriptase (Clutch), and Revert Aid RnaseH(−) RT (Thermo Scientific),were added at similar activity levels to different reaction mixturesthat each included 8 different length DNA fragments, a single sampleindex spike in DNA fragment, and 10 nM of RNA oligonucleotides. Thereactions were then carried through the entire single-stranded protocolshown in FIG. 1, but using these specific polymerases for the primerextension reaction step, 130.

The protocol outlined in Example 1 was followed, except that steps inthe primer annealing and extension section of Example 1 were modifiedwith the specific protocol for each RT enzyme as described herein.

For Superscript IV samples, Solution A was prepared by mixing theextension primer CL9 (1.38 μL, 100 μM), dNTP mix (1.6 μL, 25 mM), anddeionized water (49.04 μL). Solution B was prepared by mixing 5×Superscript IV Buffer (16 μL), DTT (4 μL, 100 mM), Rnase OUT inhibitor(4 μL), and Superscript IV RT (4 μL). Next, Solution A (26 μL) was addedto the sample. Samples were incubated at 65° C. for 2 min andimmediately placed on ice for 1 min. Next, Solution B (14 μL) was addedto the sample, and samples were incubated at 42° C. for 1 h.

For M-MLV RT (Rnase H(−)) samples, Solution A was prepared by mixingextension primer CL9 (1.38 μL, 100 μM) and deionized water (54.64 μL).Solution B was prepared by mixing 5× M-MLV RT Buffer (20 μL), dNTP mix(2 μL, 25 mM), M-MLV RT (Rnase H(−)) (4 μL), and deionized water (17.98μL). Solution A (28 μL) was added to the sample, and then samples wereincubated at 65° C. for 2 min and placed on ice for 1 min. Next,Solution B (22 μL) was added to the sample, and the samples wereincubated at 42° C. for 1 h.

For SMARTEer RT samples, 10× Reaction Buffer was prepared by mixing 10×Lysis Buffer (19 μL) and Rnase inhibitor (1 μL). Solution A was preparedby mixing 10× Reaction Buffer (1 μL) and deionized water (9.5 μL).Solution B was prepared by mixing 5× Ultra low 1st Strand Buffer (16μL), SMARTer Seq v4 oligo (4 μL, 48 μM), and Rnase OUT inhibitor (2 μL).Beads were resuspended in Solution A (21 μL). Extension primer CL9 (4μL, 12 μM) was added. Samples were incubated at 65° C. for 2 min andplaced on ice. SMARTer RT (4.4 μL) was added to Solution B, and themixture (15.0 μL) was added to the sample. Samples were incubated at 42°C. for 90 min.

For RevertAid RT samples, Solution A was prepared by mixing 4 μL 100 μMextension primer CL9, 46 μL deionized water. Solution B was prepared bymixing 16 μL 5× Thermo Reaction Buffer, 2 μL Rnase inhibitor, 3.2 μL 25mM dNTP mix, 4 μL RevertAid RT. Solution A (25 μL) was added to thesample, and samples were incubated at 65° C. for 2 min and placed onice. Solution B (15 μL) was added to the sample, and samples wereincubated at 42° C. for 1 h.

FIG. 13 provides a gel showing the products generated by the ligationmethod of Example 1 using different polymerases. Lane “F1” shows theproduct generated by Superscript IV RT. Lane “G1” shows the productgenerated by M-MLV Rnase H(−). Lane “H1” shows the product generated bySMARTer reverse transcriptase. Lane “A2” shows the product generated byRevertAid RnaseH(−).

The reverse transcriptases showed varying abilities to discriminateagainst DNA and RNA substrates the reaction mixture, FIG. 13. Lane H1shows that the SMARTer reverse transcriptase has specific activity forRNA, while the other reverse transcriptases act on both DNA and RNA.

Example 5: Concurrent Detection of Pathogens from a Low-Quality Sample

This study was carried out to test the performance of the low-qualitymethod, using cfDNA shown in FIG. 6 compared to the NuGEN Ovation®Ultralow System V2 Reagents library protocol, using double-stranded DNA.

Cell-free pathogen DNA from plasma represents a low-quality sample. Thatis, cell-free pathogen DNA is generally shorter and at much lowerconcentration than human DNA, FIG. 16. FIG. 16 shows a plot comparingthe quantity and length of cell-free DNA (measured as a function of thenumber of sequence reads) to human DNA from chr21 using high-throughputsequencing.

Briefly, blood samples were obtained from volunteers. Blood culturetests were performed in parallel on the clinical samples to confirm thatthe blood samples used contained the selected pathogens being comparedin the low-quality method and the NuGEN method.

Subsequently, high-throughput sequencing was conducted for selectedpathogens. Normalized unique reads in size-selected libraries weredetermined for the selected pathogens and the results are shown in FIG.14 and FIG. 15.

FIG. 14 shows that the ligation method detected a higher number of readsfor three of the six selected pathogens, E. aerogenes, K pneumoniae, andC. canimorsus. FIG. 15 shows that all of the selected pathogens weredetected a higher number of reads when compared with the NuGEN method,S. aureus, E. faecium, and E. coli. Furthermore, the NuGEN method failedto detect, E. coli and S. aureus in two of the infected plasma samples,FIG. 15.

Example 6: Primer Extension-Non-Templated Method Using Successive Mode(Prophetic)

A primer extension-non-templated method using a successive mode can beused to detect different nucleic acid forms in a sample, FIG. 7.

Samples can be prepared and analyzed as in Example 1 with the followingmodifications. The blunt-end repair steps in Example 1 are skipped toavoid blunting the templates. After the primer annealing and extensionsteps in Example 1, a reverse transcription step (e.g., using M-MLVreverse transcriptase or SMARTer reverse transcriptase) is introduced.

A sample having both single-stranded and double-stranded nucleic acidsis obtained and denatured to make single-stranded nucleic acids. Next, afirst adapter is ligated to the single-stranded nucleic acids. Afterligation is completed, a primer extension reaction is carried out with aDNA-dependent polymerase that has non-templated activity (e.g., Bst 2.0polymerase or the like). Following the DNA polymerase reaction, an RTpolymerase reaction is conducted that has non-templated activity (e.g.,using M-MLV reverse transcriptase, SMARTer reverse transcriptase, or thelike). Following the two primer extension reactions, a double-strandedadapter that has ends complementary to the primer-extended products isligated.

Depending on the amount of material needed in the downstream detectionassay, the products generated from the method can be amplified by PCR.

Example 7: Primer Extension-Non-Templated Method Using Concurrent Mode(Prophetic)

A primer extension-non-templated method using a concurrent mode can beused to detect different nucleic acid forms in a sample, FIG. 8.

Samples are prepared and analyzed as in Example 1 except the blunt-endrepair step is omitted.

A sample having both single-stranded and double-stranded nucleic acidsis obtained and denatured to make single-stranded nucleic acids. Next, afirst adapter is ligated to the single-stranded nucleic acids. Afterligation is completed, a primer extension reaction is carried outconcurrently with a DNA-dependent polymerase that has non-templatedactivity (e.g., Bst 2.0 polymerase or the like) and a RNA-specific DNApolymerase that has non-templated activity. After, a seconddouble-stranded adapter complementary to the primer-extended product isligated.

Depending on the amount of material needed in the downstream detectionassay, the products generated from the method can be amplified by PCR.

Example 8: Distinguishing Structural Forms of Nucleic Acids in a Sample(Prophetic)

A method for distinguishing between single-stranded and double-strandednucleic acid forms can be used to detect these different forms in asample.

This method generally, uses a dsRNA ligase and a dsDNA ligase withadapters having different identifying sequences (e.g. codes) for DNA andRNA.

Samples are prepared and analyzed as in Example 1 with the followingmodifications. Prior to the heat denaturation, a ligation step is addedusing a ligase specific for double-stranded nucleic acids (e.g., DNA orRNA). Then, end repair can be performed to generate blunt ends (FIG. 9,step 1). Next, one can use either the concurrent ligation mode orsuccessive ligation mode to attach an identifying sequence to thedouble-stranded nucleic acids in the sample (e.g. dsDNA, and dsRNA),FIG. 9, step 2. To differentiate between dsDNA and dsRNA one can use twodifferent identifying sequences in the adapters, FIG. 9, step 2. Thatis, a dsRNA ligase that attaches the adapters to double-stranded RNA canbe designed with an RNA-identifying code, and a dsRNA ligase thatattaches the adapters to dsRNA can be designed with a DNA-identifyingcode.

Next, one may proceed with the sample preparation process as providedherein, FIG. 9, step 3. Finally, the detection of the identifying codesadded to the dsDNA and dsRNA can be used to distinguish between thedouble-stranded DNA and RNA in the starting sample from thesingle-stranded nucleic acids.

In other embodiments, ligation of dsDNA and dsRNA can be performed insuccession or concurrently using ligases specific for DNA and/or RNA,such as T4 DNA Ligase. Short sequences can be deactivated to preventtheir concatemerization.

Example 9: Splint Ligase Method for Concurrent Detection of NucleicAcids in a Sample (Prophetic)

A splint ligase method can be used to detect different nucleic acidforms in a sample, FIG. 20.

Briefly, a sample can be obtained comprising both DNA and RNA nucleicacid forms. The nucleic acids in the sample are extracted and denatured.Next, a first adapter is ligated to the 3′end of RNA and DNA and the 5′ends are phosphorylated. Next, the nucleic acids are incubated with ahybrid splint molecule, composed of for example, SEQ ID NO:14 and SEQ IDNO:15, or the like, resulting in the hybridization of the splintmolecule to the 5′ of the DNA and the 5′ end of the RNA.

Next, a ligation reaction is carried out using a SplintR Ligase enzymeor the like. After the ligation reaction is completed, the sample istreated at a high temperature to release the unligated splint moleculesfrom the 5′ end of the RNA molecules. Next, one can proceed with FIG. 1,step 130. Depending on the amount of material needed in the downstreamdetection assay, the products can be amplified by PCR.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually and separately indicated to beincorporated by reference for all purposes.

1-114. (canceled)
 115. A method for performing a primer extension reaction on RNA and DNA, comprising: a) providing a sample comprising a mixture of single-stranded DNA and single-stranded RNA; b) attaching a first adapter to said single-stranded DNA, c) attaching a second adapter to said single-stranded RNA; d) annealing a first primer to said first adapter and annealing a second primer to said second adapter; e) extending said annealed first primer on said single-stranded DNA to form double-stranded DNA; and f) extending said annealed second primer on said single-stranded RNA to form a double-stranded DNA-RNA hybrid.
 116. The method of claim 115, wherein said attaching said first adapter comprises ligating said first adapter to a 3′ end of said single-stranded DNA.
 117. The method of claim 116, wherein said ligating said first adapter is performed by a ligase selected from CircLigase II, Thermostable App-DNA/RNA ligase, T4 RNA ligase 1, T4 RNA Ligase 2 truncated, and any combination thereof.
 118. The method of claim 115, wherein said attaching said second adapter comprises ligating said second adapter to a 3′ end of said single-stranded RNA.
 119. The method of claim 118, wherein said ligating said second adapter is performed using an RNA ligase.
 120. The method of claim 118, wherein said ligating said second adapter is performed using T4 RNA ligase 2 or T4 DNA ligase.
 121. The method of claim 115, wherein said single-stranded DNA is cell-free DNA.
 122. The method of claim 115, wherein said sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-alveolar lavage, urine, stool, saliva, nasal swab, and any combination thereof.
 123. The method of claim 115, wherein said extending said annealed first primer on said single-stranded DNA is performed by a DNA polymerase.
 124. The method of claim 115, wherein said extending said annealed first primer on said single-stranded DNA is performed by Bst 2.0 DNA polymerase.
 125. The method of claim 115, wherein said extending said annealed second primer on said single-stranded RNA is performed by a polymerase selected from Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, and a SMARTer reverse transcriptase.
 126. The method of claim 115, further comprising adding at least one non-templated nucleotide to a first primer extension strand.
 127. The method of claim 126, wherein said at least one non-templated nucleotide is a deoxycytidine.
 128. The method of claim 126, wherein said at least one non-templated nucleotide is added to a 3′ end.
 129. The method of claim 126, wherein said at least one non-templated nucleotide is up to eight nucleotides.
 130. The method of claim 126, wherein said at least one non-templated nucleotide is three, four, or five non-templated nucleotides.
 131. The method of claim 126, wherein said at least one non-templated nucleotide is one non-templated nucleotide.
 132. The method of claim 126, wherein said at least one non-templated nucleotide forms a first overhang.
 133. The method of claim 132, further comprising adding at least one second non-templated nucleotide to a second primer extension strand forming a second overhang.
 134. The method of claim 133, further comprising hybridizing a third adapter to said first overhang and a fourth adapter to said second overhang.
 135. The method of claim 134, further comprising sequencing said third adapter and said fourth adapter and sequences attached to said third adapter and said fourth adapter.
 136. The method of claim 134, further comprising (i) identifying sequences associated with said third adapter as originating from said DNA in said mixture of single-stranded DNA and single-stranded RNA and (ii) identifying sequences associated with said fourth adapter as originating from said RNA in said mixture of single-stranded DNA and single-stranded RNA.
 137. A method of performing an amplification reaction on a first RNA and a first DNA, comprising: a) providing a sample comprising a mixture of said first DNA and said first RNA, wherein said first DNA does not comprise a sequence complementary to said first RNA; b) tagging said first DNA with a first tag without using a transposase; c) tagging said first RNA with a second tag; d) performing an amplification or primer extension reaction on said first DNA with a polymerase that is selective for DNA templates; and e) synthesizing a complementary cDNA strand from said first RNA with a reverse transcriptase.
 138. The method of claim 137, wherein said first DNA is single-stranded DNA, double-stranded DNA, triple-stranded DNA, or a Holliday junction.
 139. The method of claim 137, wherein said first RNA is single-stranded RNA, double-stranded RNA, or a ribozyme.
 140. The method of claim 137, wherein said first DNA is cell-free DNA.
 141. The method of claim 137, wherein said sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-alveolar lavage, urine, stool, saliva, nasal swab, and any combination thereof.
 142. The method of claim 137, comprising performing said amplification to generate amplified products.
 143. The method of claim 142, further comprising sequencing said amplified products.
 144. A method of sequencing nucleic acids comprising: a) providing a sample comprising double-stranded nucleic acids and single-stranded nucleic acids; b) ligating a first adapter to an end of said double-stranded nucleic acids; c) denaturing said double-stranded nucleic acids into single-stranded nucleic acids; and d) sequencing nucleic acids ligated to said first adapter and identifying sequences associated with said first adapter as being double-stranded.
 145. A method for concurrent processing of different nucleic acid forms in a sample comprising: a) denaturing said nucleic acid forms in a sample; b) ligating a first adapter to one end of a first nucleic acid form using a ligase that has a preference for said first nucleic acid form and ligating a second adapter to one end of a second nucleic acid form using a ligase that has preference for said second nucleic acid form; c) primer extending said first and said second ligated nucleic acid forms; d) ligating a third adapter comprising a priming element; and e) amplifying said first and second nucleic forms.
 146. A reaction mixture composition comprising: a) an adapter; b) a first ligase that has a preference for a first nucleic acid form; c) a second ligase that has a preference for a second nucleic acid form; and d) a buffer.
 147. A reaction mixture comprising: a) a ligase; b) a DNA-dependent polymerase that has non-templated activity, wherein said non-templated base is N1; and c) a RT polymerase that has non-templated activity, wherein said non-templated base is N2, wherein N1 and N2 are different nucleic acid bases.
 148. A kit comprising: a) an adapter; b) a first ligase that has a preference for a first nucleic acid form; c) a second ligase that has a preference for a second nucleic acid form; and d) a buffer.
 149. A kit comprising: a) a ligase; b) a DNA-dependent polymerase that has non-templated activity, wherein the non-templated base is N1; and c) a RT polymerase that has non-templated activity, wherein the non-templated base is N2, wherein N1 and N2 are different nucleic acid bases.
 150. A method of sequencing different nucleic acids forms comprising: a) providing a sample comprising different nucleic acid forms; b) denaturing said nucleic acid forms in a sample; c) ligating a first adapter to one end of a first nucleic acid form using a ligase that has a preference for said first nucleic acid form; and ligating a second adapter to one end of a second nucleic acid form using a ligase that has preference for said second nucleic acid form, wherein said first and said second adapter comprise different identifying sequences; and d) sequencing said ligated nucleic acids, thereby identifying said different nucleic acid forms in said sample.
 151. A method for processing different nucleic acid forms in a sample comprising: a) denaturing said different nucleic acid forms in a sample, wherein said different nucleic acid forms comprise a first nucleic acid form and a second nucleic acid form; b) attaching a first adapter to said first nucleic acid form and a second adapter to said second nucleic acid form; c) amplifying said first nucleic acid form using a DNA-dependent polymerase that has non-templated activity, wherein said non-templated activity comprises adding at least one N1 nucleotide or a first sequence to amplified products of said amplification of said first nucleic acid form; and d) amplifying said second nucleic acid form using a reverse transciptase polymerase that has non-templated activity, wherein said non-templated activity comprises adding at least one N2 nucloetide or a second sequence to amplified products of said amplification of said second nucleic acid form, wherein said N1 nucleotide and said N2 nucleotide are different nucleotides or said first sequence is different from said second sequence. 